Appendix 1: Confocal Microscopes
range of confocal microscopes is available - from the highly versatile but very expensive "top end" A instruments, to specialised high-speed imaging and even miniaturised medical instruments. Confocal microscopes are at the forefront of the upsurge in interest in light microscopy in the past 15 years. The technology is still evolving fast - so you should expect major changes and innovations in the coming years. This appendix gives an overview of the hardware for most of the laser scanning confocal microscopes currently available. Although not all manufacturers are described in as much detail as others, this does not in any way reflect on the instruments - this imbalance simply reflects the difficulties in compiling the necessary information for each of the instruments in the time available. I hope that this appendix is an evolving project that will include not only updated, but also expanded information on each ofthe instruments in subsequent editions ofthis book. Laser Spot Scanning Confocal Microscopes These instruments are based on the use of a finely focussed laser "spot" that is scanned across the sampie. The retuming fluorescent or backscattered light is directed through a confocal iris or pinhole that eliminates out-of-focus light. BioRad Cell Science Division ______--'pO<:a..,g.."ec..::3=56
CarIZeiss______p~a~gce~3~8~2
Leica Microsystems______---"p~a!.llg~e..:!:4=02
Nikon______~p~aAg~e~4~15
Olympus______~p~a~gce~4~2~O Nipkow Spinning Disk Confocal Microscopes The Nipkow disk contains a large number of fine "pinholes" arranged in a spiral array such that a confocal image is created when the disk is spinning. These instruments operate at higher speeds than a laser single spot scanning system. They also result in less intense light irradiation at each spot on the sampie, significantly reducing fluorophore fading and cellular damage.
Atto Bioscience______~p~a~gce~4""3:..:.1 Nipkow Disk / Micro-Iens Array Confocal Microscopes A further modification of the basic Nipkow disk confocal microscope is the incorporation of a second disk, containing an array of micro-lenses, that focuses the laser light through the pinholes on the Nipkow disko Compact and robust micro-lens array Nipkow disk scan heads (CSUlO and CSU2l) are produced by Yokogawa Electric Corporation, and are assembled into confocal microscopes by a number of manufacturers.
Yokogawa Electric Corporation ______...... l!.p~agt&:e~4~3~5
Confocal Microscopes using the Yokogawa Scan Head
PerkinElmer______~pa~g~e~4~3~8
VisiTech International, ______r:;pa""g~e'_'4""4'"'"1
ConJocal Microscopy Jor Biologisls. Alan R. Hibbs. Kluwer Academic ! Plenum Publishers, New York, 2004. 355 356 Bio-Rad Bio-Rad Cell Science Division
he Bio-Rad laser scanning confocal microscopes are capable of superb resolution and have enormous T versatility for multiple fluorescence and transmission imaging. This appendix is aimed at giving an overview of the hardware involved in the Bio-Rad confocal and multi-photon microscopes. Although the latest confocal microscopes from Bio-Rad are almost fully controlled from the computer, a basic understanding of the physical design of a confocal microscope scanning system is important for both getting the most out of your instrument and rninimising your frustrations when learning to use the software. This appendix explains in some detail the various hardware components of the Bio-Rad Radiance and MRC confocal microscopes. For more detailed information please refer to the Bio-Rad instrument technical and user manuals.
BIO-RAD INSTRUMENTS The latest Bio-Rad confocal rnicroscopes (the Radiance series) have a relatively small scan head that can be readily transferred from one rnicroscope to another (Figure A-l). The small and compact scan head is factory aligned, and so no maintenance or alignment is required once the instrument is installed. The scan head itself will need to be re-aligned into the microscope if you do move the scan head from one microscope to another. This is not a difficult task and can be readily accomplished in a few minutes. Bio-Rad is a specialist manufacturer and innovative developer of the laser scanning confocal microscope scan head, associated optics, electronic components and computer software for controlling the microscope. Bio-Rad laser scanning confocal microscopes are manufactured in the UK, with a major sales distribution and technical support division in the USA. Bio-Rad, unlike a number of the other laser scanning confocal microscope manufacturers, is not an optics or microscope manufacturer. The Bio-Rad scan head can be fitted on microscopes from a number of manufacturers, but in more recent years Bio-Rad has favoured selling a complete package that inc1udes a research grade light Figure A-l. Bio-Rad Radiance Confocal Microscope. microscope from Nikon. The small Radiance scan head is shown attached to the video port of a Nikon upright microseope. The associated controller box, laser and computer are not shown in this photo. (This figure is adapted from a photo graph kindly provided by Bio• Rad Cell Science, UK).
Bio -Rad Cell Seien ce Division Web' htto-Jlcellscience biorad corni Address : Bio-Rad House Life Sciences Group. Unit I block Y Maylands Avenue, Heme! Hempstead 2000 Alfred Nobel Drive Regents Park Industrial Estate Hertfordshire, HP 7TD Hercules, CA 94547 391 Park Rd, Regents Park NSW 2143 UK USA Australia Phone : +44 (0) 208 328 2141 +1 (510) 7416868 +61 299142800
FAX : +44 (0) 208 223 2500 +1 (510)741 5811 +61299142888 Appendix I: Confocal Microseopes Bio-Rad 357
Bio-Rad Confocal Microscopes
Radiance Confocal Microscopes Bio-Rad Cell Science Division is a specialist Fully computer controlled small and robust scan head. with associated manufacturer of confocal microscopes. This JCU (Instrument Control Unit). which is located under the work bench. The scan head can be readily moved between an upright and an inverted includes their unique scan head and controller microscope. and can be mounted on a number 0/ different light box design, which can be readily attached to a microscopes by using the correct adaptor. number of microscopes. Bio-Rad is not a Radiance 21 OOTM Spectra1 imaging version of the Radiance 2100 manufacturer of microscopes or lenses. Rainbow confoca1 microscope (can also be installed as an upgrade to an existing Radiance 2100 Bio-Rad first introduced biologists to confocal instrument). See Figure 3-14 (page 85). microscopy in the late 1980's with the MRC- 500. This was later upgraded to the MRC-600, Radiance 21 OOTM Single, dual or tripIe channel, fully computer controlled confocal microscope using Windows MRC-lOOO and subsequently the MRC-I024. NT/2000 LaserSharp software. Can also be These upgrades provided greatly increased purchased multi-photon ready. sensitivity, further computer control of the Radiance 21 OOTM MP Multi-photon combined with a confocal, or microscope and a much more versatile multi-photon dedicated (MPD). See Figure 3-19 instrument. The MRC-I024 has the capability (page 94). of tripie fluorescence and tripie channel CellMapTMID "Personal" confocal microscope with 405nm and transmission imaging simultaneously. A 488 nm solid state laser confocal microscope for completely redesigned confocal microscope, DAPIICFP and GFPIFITC. (the Radiance series of confocal microscopes) CellMapTMIC "Personal" confocal microscope with 488nm & has now replaced the MRC series. 532 nm solid state lasers designed for GFPIFITC/TRITC/Cy3. The Bio-Rad confocal microscopes have the unique characteristic of using infinity optics Micro Radiance™ Same optical design as the Radiance, but with within the scan head (parallel light beams) that more Iimited versatility. allows the use of a large (mm size) variable RTS2000 Fully computerised 3 channel high-speed video- iris to function as the pinhole. This has the rate imaging using aresonant galvanometer. advantage that alignment of the laser and scan RTS2000MP Multi-photon version video-rate instrument. head is very robust and greatly simplified. Due to this simplicity of scan head alignment, the Earlier MRC Confocal Microscopes The large scan head 0/ the MRC series houses all 0/ the optics and the Bio-Rad confocal microscope scan heads can P MT tubes. These instruments are no longer manu/actured, but continue be installed on many different conventional to be supported technically by Bio-Rad where possible. light microscopes. MRC-1024 Most scan head optical components controlled A significant change to the Bio-Rad confocal via the computer using LaserSharp software, microscopes in more recent years, as far as the originally operating under OS/2, but can be upgraded to run under Windows NT. Tripie use is concerned, has come about due to the fluorescence and tripie transmission irnaging. reliability and cheapness of quite powerful MRC-1024MP Multi-photon version of the MRC-1024 confocal computers. This has resulted in very microscope. significant change in the microscope user interface, where the instrument is now MRC-lOOO Multi-channel confocal microscope with improved sensitivity compared to MRC-600. completely controlled by the computer.
MRC-600 Significantly more sensitive fluorescence The development of the Bio-Rad laser detection than the MRC-500. Improved COMOS scanning confocal microscopes has software running under DOS. traditionally been closely associated with the MRC-500 Original commercially available laser scanning academic research environment, and many confocal microscope from Bio-Rad, with settings innovative changes have come through on the scan head being manually controlled. Images collected using SOM software under feedback from customers all over the world. DOS.
Appendix I: Confocal Microscopes 358 Bio-Rad
RADIANCE SERIES OF CONFOCAL MICROSCOPES The Radiance series of confocal microscopes from Bio-Rad are based on a relatively small factory aligned scan head, which is attached by fibreoptic cable to a controller box, which houses the lasers, optical filters and PMT detectors (Figure A-2). This simple modular dcsign means that various components can be readily upgraded or further optical filters or lasers added without the need to replace the scan head. This section describes in some detail the design of the Radiance series of confocal microscopes, with particular emphasis on the Radiance 2100 instrument. Radiance 2100: is a high-end confocal microscope based on the use of dichroic mirrors and optical filters to separate the fluorescent light into various regions of the light spectrum. The instrument is capable of simultaneous tripie labelling, transmission and backscatter (reflectance) imaging. Instrument settings are all computer controlled via the LaserSharp software running under Windows NT/2000. The Radiance 2100 Rainbow confocal microscope is capable of separating fluorophores with highly overlapping emission spectra by spectral analysis using aseries of computer controlled optical filters.
LSS8rShatp conlrols Ihe microscope DichroIc mirrors separate difrerent waveleng/IIs. and Ihe variable size iris (confocal 'pinhole") ",molleS "out of Control foaJs· flUOfBScence. panel InvertId
Instrument ., Control r
Unit o (ICU) Computer The Image is digitised wffhin Ihe controller box, and then Ir8nsferred to Ihe computer Laser LJ
Figure A-2. Radiance Confocal Microscope. The Radiance confocal microscopes from Bio-Rad have a relatively small scan head that can be readily moved from one light microscope to another. Furthermore, the scan head is factory aligned and all that is required from the user is for the scan head alignment into the microscope to be adjusted. Most of the optical components, PMTs, lasers etc are located in the Instrument Control Unit (ICU) that is located below thc microscope bench.
Appendix I: Confocal Microscopes Bio-Rad 359
Components of the Radiance Confocal Microscopes The Radianee laser spot seanning eonfoeal microseopes ean be eonfigured for visible light and multi• photon mieroseopy on either upright or inverted light mieroseopes. Fluorescent light separation is based on dichroie mirrors and optieal filters. The small scan head is direetly attaehed to a light microseope, with the deteetion opties and lasers housed in a remote unit. The o instrument is a high-end eonfoeal mieroseope that has great versatility. Perrnanently faetory aligned small and robust sean head ean be easily moved between mieroseopes. Polarisation beamsplitter: The primary beam splitter (for separating irradiating laser light from o fluoreseent light) is based on polarisation - a very efficient means of collecting the fluorescent light. Dichroic mirrors: Mounted inside the sean head is a eomputer-controlled wheel that contains a number of dichroic mirrors that are used to provide an initial separation of the fluoreseence emission. Variable size confocal iris: A computer controlled variable size "pinhole" or iris is mounted in front of eaeh fibreoptic piekup (one for each channeI) within the scan head. The scan head optics is designed such that the confoeal "pinhole" is relatively large (mm size), which greatly simplifies alignment. Fibreoptic pickup: The fluoreseent light, after passing or being defleeted by suitable diehroie mirrors, is transmitted to the controller box by multi-mode fihreoptie eable. Scanning mirrors: X & Y fast galvo sean mirrors with two assoeiated eoneave mirrors to ensure the back aperture ofthe objeetive is fully filled throughout the sean. Zoom, bi-direetional & sean rotation. SEL (Signal Enhancing Lens): A removable lens system that is used to direet more out-of-foeus light to the deteetors (greatly inereasing instrument sensitivity) with only a smallioss in x-y resolution. Modular Instrument Control Unit (lCU) eontains most of the optieal eomponents. Built in a modular way such that individual components can be easily replaeed or upgraded. The scan head is conneeted to the controller box via a large diameter multi-mode fibreoptie connection. Barrier filters: A computer-eontrolled wheel of removable barrier filters is mounted in front of each of the PMT tubes. Spectral imaging is available on the Rainbow model by using aseries of computer controlled Long Pass and Short Pass interferenee filters. Light detectors: Prism enhanced photomultiplier tube for each channe!. Lasers: A wide range of lasers can be attached via fibreoptic connection, including the Kr/Ar, Ar-ion, HeNe, 405 nm violet, red laser diode and 561 yellow laser diode, HeCd and single line Kr-ion. Ti:sapphire multi-photon lasers ean be attaehed to suitably configured instruments. Laser attenuation: Computer selected ND or AOTF filters loeated within the Instrument Control Unit. Laser line selection: Laser line se!eetion is controlled by computer eontrolled optical filters or AOTF filters loeated in front of eaeh laser, prior to fibreoptic cable transfer to the scan head. The relative!y small scan head can be easily transferred between an upright or inverted microscope. The Bio-Rad scan head can be mounted onto a variety of research grade light microseopes from different manufacturers. Bio-Rad is not a manufaeturer of light microseopes or lenses . Multi-ehannel fluoreseenee (up to 3 ehannels), photon eounting, backscatter (reflectance), transmission. • Box sizes user seleetable up to 1280 x 1024 pixels. Line, ROr, zoom, panning and rotated seanning. Simultaneous and sequential multi-ehannel seanning. Emission ratio and mixer eombinations. 12-bit data aequisition and proeessing, saved as 8-bit or 12-bit image files. Time lapse imaging. '""d ~TIwBmiIIIDD Single ehannel photodiode deteetor or PMT deteetor (with additional filters). Capable of bright-field, phase and DIC imaging, depending on the optieal settings available on the light microseope. Programmable manual control unit can be used to control all ofthe essential acquisition parameters. High-end Windows computer eontrols the confoeal mieroseope and eolleets the images. A single large computer sereen or a dual display eonfiguration is possible. 111ft LaserSharp aequisition and analysis running under Windows NT/2000 eontrols all seanning assoeiated funetions. Also used for some image manipulation, eoloealisation and 3D reeonstruetion. Bio-Rad also seils aseparate 2D image analysis program (LaserPix) and 3D visualisation and measurement software (LaserVox). Confoeal Assistant (free software available from Bio-Rad; see page 168 ) is a handy program for basic image viewing and manipulation.
~"""* Multi channel (up to 3 fluoreseence + I transmission), single ehannel system ean be upgraded to multi- .... _-....;..= ehanne! after purehase. Multi-photon (page 94), with optional external deteetors. High-speed video• rate resonant scanning system. Spectral imaging is available on the Rainbow instrument (page 83).
Appendix I: Confüeal Microseopes 360 Bio-Rad
Radiance Scan Head The Radiance scan head (Figure A-3) contains the scanning mirrors, the polarisation beamsplitter for separating the irradiating laser light from the retuming fluorescent light, the dichroie mirrors for separating the different wavelengths of fluorescent light, the variable iris and a fibreoptic pickup port for transmitting the light to the Instrument Control Unit. All other optical components are housed within the Instrument Control Unit that is usually located under the microscope bench (Figure A-4). The operation ofthe Radiance scan head is fully controlled by the Bio-Rad produced LaserSharp software. The x-y galvometric mirrors located within the Radiance scan head, are factory aligned and cannot be adjusted by the user. The relatively small and compact nature of the Radiance scan head makes this scan head particularly easy to transfer from one computer to another. There are only minor DIchroIe mlrrors 'CM' alignments to be done when the 2 chann" Mlcro Radla.nc. scan head is moved to another 1 100% DEr1 microscope. This inc1udes both 2560DCLP 3. 100% D_ET_2__ -, lateral adjustment of the scan head and beam focus adjustments. The scan head is connected to the associated Instrument Control Unit via a large diameter flexible metal tubing that houses the control cables and fibreoptic cables for each channel. The fast galvo scanning mirrors in the Radiance 2100 confocal microscope are designed such that the scan can be accomplished in any direction. This means that a narrow scan can be executed at an angle across the screen. This is useful, for example, when scanning an individual neuron presented at an angle across the sampIe. The scanning mirrors in the earlier Micro Radiance instruments cannot be "angled" under OS/2, but angled scanning IS possible if these instruments are upgraded to Windows 2000. Two concave mirrors, located on either side of the x and y galvo scan mirrors (shown in Figure A-3), provide a scanning geometry that ensures that the back aperture of the objective is filled throughout the scan cyc1e (this is important for maximising the optical resolution of the instrument). Figure A-3. Radiance Scan Head. Fluorescent and reflected light The Radiance series of confocal microseopes has a relatively small and robust scan retuming from the sampIe is de• head. Many optical components, previously housed in the scan head, have now been scanned by passing back through transferred to the Instrument Control Unit (ICU), see Figure A-4. This figure, adapted the scanning mirrors. The light is from a figure kindly provided by Bio-Rad, UK, is of a dual channel Micro Radiance confocal microscope. then transmitted through a polarisation beam splitter to a Appendix I: Confocal Microseopes Bio-Rad 361 telescope lens arrangement (allowing this scan head to be much smaller than the previous MRC models). This lens arrangement is necessary to obtain the correct optical configuration when aligning the light through the variable pinholes. The polarisation beam splitter is used to separate the highly polarised irradiating laser beam from the essentially randomly polarised retuming fluorescent light. This type of polarisation beam splitter, prevents any fluorescent light that is polarised in the same direction as the irradiating laser from entering the detectors, but does allow the full range of the light spectrum to be detected. Unlike a dichroie mirror, there are no regions of the spectrum where light cannot penetrate the beamsplitter. A computerised wheel containing a number of dichroie mirrors is used to split the fluorescent light into the relevant component wavelengths. In the two channel Micro Radiance scan head (shown in Figure A-3) there is a
PMT2
~ Q) U) PMT1 CO -..J C o Q) Amplifier -...Z Digitiser Fibre optic cables E from scan head :::s Neutral density filterwheel Frame grabber Q) Une selectOf I Output J t to compufe, Argon Laser
Figure A-4. Radiance Instrument Control Unit (I CU). The Radianee Instrument Control Unit now houses the lasers (including the laser anenuation neutral density or AOTF filters and laser line seleetor optieal filter wheels), the PMT tubes for deteeting fluoreseent light (including the baITier filter wheel), the amplifier, digitiser and frame grabber. The digitised signal is then transfeITed to the computer for proeessing.
Appendix I: Confoeal Microseopes 362 Bio-Rad
single dichroic mirror wheel. In the Radiance 2100, which is capable of simultaneous tripie labelling, there are two wheels of computer controlled dichroic mirrors. After suitable reflection or transmission via the dichroic mirror the light is aligned into the variable iris mounted in front of the fibreoplic cable pickup ports. Due to the infinity optics design of the scan head the "pinhole" is relatively large, being mrn in diameter and circular in shape. The fluorescent light is transferred to the Instrument Control Unit via a large diameter fibreoptic cable to minimise loss oflight. The large diameter multi-mode fibre also means that the optic fibre does not act as a "pinhole", and hence the requirement for the variable iris or confocal "pinholes" mounted within the scan head. Radiance Instrument Control Unit (ICU) Many of the optical components previously housed in the scan head have been transferred to the Instrument Control Unit in the Radiance series of confocal microscopes (Figure A-4). This has been made Filters - detector 1 possible by using large diameter multi-mode fibreoptic cables between the scan head and the Instrument Control Unit as discussed above. 1. OPEN Mounted within the Instrument Control Unit are the lasers and their power supplies. The argon-ion or krypton-argon-ion lasers require a 2. ES70LP cooling fan that is mounted outside the Instrument Control Unit, but the 3.E600LP helium-neon lasers do not generate excessive heat and so do not need an extemal-cooling fan. The argon-ion I krypton-argon-ion laser cooling fan is shut off automatically when the laser has cooled sufficiently (approximately 5 minutes) after the laser unit has been tumed off. The power supply to the Instrument Control Unit should be switched to "standby" when an imaging session has been completed, to ensure the fan will remain on until the laser has cooled. Computer controlled neutral density filters and line selection filters (Figure A-4), or computer controlled AOTF filters are used to attenuate the amount of laser light that enters the microscope and to select individual laser lines. These filters are mounted in front of each ofthe installed lasers. The laser light, after passing through the attenuation and line selection filters is then picked up by fibreoptic cable, combined into a single fibreoptic cable, and transferred to the scan head. Large diameter multi-mode fibreoptic cables are then used to transfer the fluorescent or reflected light from the scan head back into the Instrument Control Unit (with aseparate fibreoptic cable for each detector channei). There are separate computer controlled barrier filter wheels mounted in front of each ofthe PMT tubes within the Instrument Control Unit to further define the region of the light spectrum that will be detected by each individual channel. For specialised applications the barrier filters loaded in the filter wheels can be changed. Additional optical filter wheels are mounted inside the Instrument Control Unit of the Radiance 2100 Rainbow confocal microscope for Filters - detector 2 spectral imaging (see Figure 3-14 on page 85). A combination of Long Pass and Short Pass sharp cut-off interference filters are used to dissect 1. OPEN the visible light spectrum into narrow bands of light. Automated optical 2. HOSOOLP filter selection is used to generate aseries of images that make up a spectral scan of the sampie. Using these optical filter wheels for both 3. HOS1S/30 spectral imaging and spectral reassignment in multi-Iabelling imaging 4. HQS30/60 applications is discussed in detail below. A prism enhanced PMT tube (a more efficient light collector compared to a conventional PMT tube) is used foreach individual detection channel. The output from the PMT tubes is amplified and then digitised within the Instrument Control Unit, before transfer to the computer for display and further processing. The modular nature and large size of the Instrument Control Unit now makes the replacement of defective components or the upgrading of individual components a relatively simple task.
Appendix 1: Confocal Microseopes Bio-Rad 363
Changing Optical Filters and Dichroic mirrors A number of dichroic mirrors (in the scan head) and barrier filters (in the Instrument Control Unit) can be mounted within the computer controlled filter wheel and so there should be little need to consider changing the filters. However, these optical filters and dichroic mirrors can be changed if necessary. The filter is designed to slip out by holding the loop at the top ofthe filter holder. Another filter can then be placed in the empty position (see the instrument manual). Y ou will then need to update the information in the LaserSharp software to reflect the new filters that have been installed. Radiance 2100 Rainbow The Rainbow version of the Radiance confocal microscope uses two computer controlled interference filter wheels (see page 85) to separate fluorescent light into separate regions of the light spectrum. One optical filter wheel has a set of short-pass interference filters and the other a set of long-pass filters. In combination these two optical filter wheels can be used to select a defined region ofthe light spectrum. Each ofthe fluorescence imaging channels (up to 3) available on the Radiance 2100 Rainbow confocal microscope has a separate dual filter wheel assembly, pinhole and PMT detector. The dual optical filter wheel assembly in the Rainbow is a very convenient method of separating regions of the light spectrum, with the Radiance 2100 Rainbow flexibility of allowing one to choose individual Spectral separation of fluorophores using computer regions to be directed to each detector. The filter controlled interference filters (see page 85). wheels not only provide considerable flexibility when choosing spectral regions, but they also provide an intuitive method of choosing the correct optical filter. The on-screen filter display is listed as aseries of optical filters on a drop down menu (the blocking filter set and the emission filter set - see page 380). Each optical filter in the series has a 10 nm difference in the spectral cut -off point, and as the filters are arranged in sequential order it is relatively easy to choose the correct filter. Furthermore, Bio-Rad now provide a visual on-screen spectral diagram that includes a coloured outline ofthe part ofthe light spectrum directed to each channel. The dual filter wheel assembly in the Rainbow instrument can also be used to obtain spectral information from the sampie. A spectral image series or lambda stack is generated by collecting aseries of images, each from a narrow region of the light spectrum (as narrow as 10 nm if required). The spectral image series can be used to study spectral changes within the cell or tissue sampie, or for spectral separation of individual fluorophores. Spectral information can also be used, in a process called spectral reassignment, to separate probes with highly overlapping emission spectra. If fluorescent probes with overlapping emission spectra are imaged using two or more collection channels then there will be bleed-through between the channels. On Bio-Rad instruments a small amount of bleed-through can be corrected while imaging by subtracting a small percentage of one collection channel from the other collection channel using on-screen mixers. However, when the bleed-through is more substantial, and is occurring in more than one channel then the process of spectral reassignment is more appropriate. This process is similar to the Zeiss META spectral unmixing algorithm, but is performed on only two or possibly three spectrally separated images rather than a lambda stack. This has the advantage of very fast simultaneous collection of the spectral information. The degree of bleed-through between individual channels can be calculated from images collected using single labelIed control sampies. In the Bio-Rad spectral reassignment process the light from defined regions ofthe light spectrum is directed to a individual (single PMT) detection channels. This greatly increases the sensitivity of the instrument, and also allows one to easily adjust the relative intensity of the signal for each of the channels. For spectral reassignment to be successful both fluorophores must show similar intensity in the image. Adjusting the individual collection channels is readily accomplished by changing the gain for each of the detection channels. The Bio-Rad method of spectral separation does have the advantage of increased sensitivity and speed of acquisition and analysis, but under conditions of high levels of fluorescence the Zeiss META linear unmixing algorithm used on a full lambda stack may be able to separate a larger number of fluorescent probes.
Appendix 1: Confocal Microseopes 364 Bio-Rad
SELS (Signal Enhancing Lens) The Signal Enhancing Lens (SELS) is a small removable optical device that greatly increases the amount of out• of-focus light that reaches the detectors without greatly reducing the x-y resolution of the instrument. Inserting the SELS lens reduces the effective magnification of the light from the Radiance telescope lens, resulting in more light passing through the confocal pinhole or iris without the need to greatly increase the size of the iris. The greatly increased signal provided by the SELS lens is ideal for live cell imaging where photobleaching and photo damage often seriously limit the time allowed for following cellular events in live cells (see "SELS: A New Method for Laser Scanning Microscopy of Live Cells by Reichelt and Amos, Microscopy and Analysis, November 2001 - further details in Chapter 15 "Further Reading", page 350). Photon Counting Bio-Rad confocal microscopes are capable of "photon counting", where electronic circuitry is used to "count" each photon as it is collected by the photomultiplier tube (see page 37 and 110). Photon counting is only applicable when using sampIes with low levels of fluorescence. Excellent images, with low noise levels, can be collected with photon counting if the "accumulate" mode is used when collecting several images. When using the photon counting mode an image may not be visible with a single scan - a number of sc ans may need to be accumulated before the image becomes visible. Photon counting can also be utilised on the Bio-Rad Radiance Rainbow instrument when collecting multi• channel images for the separation of highly overlapping emission spectra using spectral reassignment. Computer and Software A DELL IBM compatible computer is used to operate the Radiance series of confocal microscopes. Only a relatively modest computer is required for operating the confocal microscope and collecting images (sufficient to run Windows XP or NT), however, 3D image processing will be smoother and faster with a computer with a large amount of memory (RAM) a large hard disk and a fast video card. The Micro Radiance series of confocal microscopes are operated by using either the older version of LaserSharp, which runs under OS/2, or utilising the new version of LaserSharp that runs under Windows NT. Confocal microscopes still operating under the OS/2 operating system can be upgraded to the latest Windows operating system. However, as this requires the installation ofboth the operating system and new software for controlling the microscope, don't attempt an upgrade unless you are familiar with making such major changes to a computer! The LaserSharp software is designed to control the confocal microscope, collect images and to perform most elementary image processing. This includes 3D reconstruction and colocalisation algorithms. The LaserSharp software is discussed in more detail later in this appendix (page 377). The spectral reassignment algorithm is available on the Radiance 2100 Rainbow confocal microscope using SpectraSharp.
Appendix 1: Confocal Microscopes Bio-Rad 365
MRC SERIES OF CONFOCAL MICROSCOPES The MRC series of confocal microscopes, from the MRC-500, which was one of the earliest confocal microscopes used extensively in the biological sciences (first introduced in the early 1980's) to the highly versatile MRC-I024 (produced up to 1998) are used in a large number of Universities and Research Centres throughout the world.
Fibre optic connection ~ Laser
DIdvDic mIfrors~" dt""'- WlJ\/lJl8ngIhs. lind /fIe varisb18 si.e iIIs rPirilole' ItIm = = Controller = o o box o The Image ;s digitised with./n I/Ie conIIoIIer box. end IhM It'8nSIemKllO I/Ie computer r; Figure A-5. MRC-1024. The MRC series of confocal microscopes have significantly larger scan heads compared to the Radiance confocal microscopes. This is due to most of the optical components, and the PMT tubes being located in the scan head. The MRC scan head can be mounted on both upright and inverted research grade microscopes !Tom a variety of microscope manufacturers. Although the MRC instruments are now superseded by the Radiance confocal microscope design (see previous section), they are still excellent instruments with many still in operation - and continue to be technically supported by Bio-Rad. In this section a detailed description is given of the latest in the MRC series, the MRC-1024 confocal microscope. The previous members of the MRC confocal microscopes (MRC-500, MRC-600 and MRC-IOOO) are based on the same fundamental design used in the MRC-1024. The major difference between these earlier model MRC instruments and the MRC-I024, as far as the user is concemed, is the extensive use of computer software in the latter to control the settings of the confocal microscope scan head. There are also many important technical advances utilized in the MRC-I024 instrument that make this confocal microscope significantly more sensitive and more versatile than the earlier MRC instruments. The LaserSharp software, used for controlling the new Radiance confocal microscopes, can also be implemented on the MRC-I024 and MRC-IOOO, but the earlier versions of the MRC confocal microscope (MRC-500/600) are controlled by the DOS based COMOS software. Appendix I: Confocal Microscopes 366 Bio-Rad Components ofthe MRC-1024 Confocal Microscope Tbe MRC-I024 laser spot scanning confocal microscope can be configured for visible light, UV light and multi-photon microscopy on either upright or inverted light microscopes. DeIIp A large scan head, which houses all of the necessary optics, pinholes and detectors is direcdy attached to an upright or inverted microscope. Tbe instrument is computer controlled, with the exception of the changeover of specific optical filter blocks in the scan head. Sc-. Had Tbe relatively large and heavy scan head of the MRC series of confocal microscopes means that mounting the scan head on another microscope is difficult - even though Bio-Rad advertised this instrument as being readily moved from one microscope to another. The MRC-I024 scan head contains all of the necessary optics and PMT tubes for multiple channel labelling. As a consequence the scan head is not only rather large, but also a delicate instrument. Filter blocks: Removable optical filter and dichroic mirror blocks are housed within the scan head. Changing the laser may require a different combination of optical filter blocks. Dichroic mirrors: Dichroic mirrors for specific applications are changed by substituting different ('-l optical filter blocks. Variable size confocal iris: A variable size iris, or "pinhole", is mounted in front of each PMT tube and its size is under computer contro!. Light detectors: Three photomultiplier tubes (PMT), allowing for 3 colour simultaneous fluorescence 0 imaging, are housed within the scan head. Scanning mirrors: X & Y fast galvo scan mirrors. Zoom and bi-directional capability. C...... BIII The image is digitised within the controller box and then transferred to the computer. The transmission image (3 channels in the MRC-1024) is also detected within the controller box, after collection on the microscope and transfer to the controller box by a fibreoptic cable. Lasers: A wide range of lasers can be attached via fibreoptic connection, including the Kr/Ar, Ar-ion, HeNe, red laser diode, HeCd and single !ine Kr-ion. UV and Ti:sapphire multi-photon lasers can be I attached to suitably configured instruments. Laser attenuation: Computer selected ND or AOTF filters located within the laser box. Laser line selection: Computer controlled filters for laser line selection are located in front of each laser, prior to fibreoptic cable transfer to the scan head. The scan head can be attached to either an upright or inverted microscope, but moving the scan head to U another mieroscope is somewhat diffieult due to the relatively large size and delieate nature of the scan head. Bio-Rad has designed adaptors for the MRC-I024 sean head to be attaehed to a research grade light microscope from each ofthe major microscope manufacturers. Multi-channel fluorescenee, photon counting, backscatter (reflectance), transmission. Box sizes user seleetable up to 1280 x 1024 pixels, zoom and panning. Simultaneous and sequential multi-channel seanning. Mixer eombinations. 12-bit data acquisition and processing, saved as 8-bit or 12-bit image ~ files. Time lapse imaging. 1'1_ 64 • A mirror located after the condenser (above the condenser on an inverted mieroscope) is used to direct transmitted light to up to three detectors located within the controller box. The operation of the transmission detectors is via the LaserSharp software. However, a mirror on the microscope must be manually changed to direct the laser light into the fibreoptic pickup cable. Programmable manual control unit is not available for the MRC instruments. High-end PC computer with a single large computer screen to display the images as they are collected ~ and to display the controls for scanning and changing microscope settings. More complex image analysis is usually done using software installed on a separate computer. LaserSharp acquisition and analysis, running originally under OS/2 but can be upgraded to run under Windows NT, controls all scanning associated functions. Also used for some image manipulation and 3D reconstruction. Bio-Rad also seils aseparate 2D image analysis program (LaserPix) and 3D visualisation and measurement software (LaserVox). Confocal Assistant (free software available from Bio-Rad; see page 168) is a handy program for basic image viewing and manipulation. The MRC confocal microscopes are no longer manufactured, but multi channel (3 fluorescence + 3 transmission), multi-photon and UV systems are still in use around the world. Appendix I: Confocal Microscopes Bio-Rad 367 MRC-1024 Scan Head The Bio-Rad MRC-1024 scan head (Figure A-6) contains an extended light path that is folded back several times (to gain sufficient length for optical requirements without using a long optical box). The scan head contains all of the optical components, a variable iris for each channel and the photomultiplier (PMT) tubes. This results in a rather large and delicate scan head that, although it can be attached to both upright and inverted microscopes, is not easily transferred from one microscope to another. The new Radiance series of confocal microscopes from Bio-Rad has a radically "down sized" scan head, where many optical components have been transferred to the Instrument Control Unit (discussed in detail in the previous section), and the light path has been significantly shortened by using a telescope lens. Adjustable mirror Flpre A~. MRC-I014 Sen Und. Tbc MRC·1024 scan be~ CO!I.tains IhRe photomultiplier rubes (pMT I, PMTI, PMTI), and IWO removable dichroie mirror sets (D I and 02). MI, M4 and M5 Ire user ~justable mirrors 10 aJlow one to corm:tly aJign the laser beam into the PMT rubes. Minors M2. M3 and M6 Ire fllClory set. Tbc scan b~ also contains computer controllcd opticaJ filter wbcel for refining the wlvelengths of light lbat will be detectcd by eacb of the PMT rubes. Filter Block Position 1 ~ttM- Laser beam entry Adjustable mirror • M1 • M3 Adjustable mirror Microscope Appendix 1: Confocal Microseopes 368 Bio-Rad There are a number of mirrors positioned within the MRC-l 024 scan head for the purpose of both aligning the laser light into the objective on the microseope, as weil as aligning the retuming fluorescence light into the iris and PMT tubes (denoted MI to M6 in Figure A-6). A number ofthese mirrors are factory aligned (M2, M3 and M6) but some of these mirrors are user adjustable (M I, M4 and M5). Extreme care should always be taken when attempting to adjust these mirrors, as incorrect alignment of the mirrors can cause considerable loss of sensitivity and uneven detection across the field ofview. The Bio-Rad confocal microscope scan head can be fitted to a variety of light microseopes from a number of manufacturers. This includes both upright and inverted microscopes. The scan head is best connected to the microscope as close to the objective as possible (to minimise problems with light loss from the many optical components within La er the microseope). Less sensitivity than usual? r'V When the scan Alignnrellt head is mounted on Try re-aligning the scan head mirrors: an inverted MI, aligns the laser beam into the scan head. Use the microscope the coupling is via an attachment "bulls eye" prism, provided by Bio-Rad. that allows the laser to be delivered directly to the objective. This means that the confocal M4, careful adjustment of M4 will align the fluorescent microscope image quality obtained on an light into PMTl. Poor alignment results in low signal and inverted microscope is exactly the same as that uneven lighting. obtained on an upright microscope when using M5, this mirror allows you to align the fluorescent light the same objectives. However, the scan head can into PMT2. be mounted in a variety of places, including the See Figure A-610r location 01 alignment mirrors. video mount on the top front of an inverted microscope, with only a small loss in image quality. Scan Head Alignment The alignment of the scan head with the microscope is most important for high quality imaging. Although the scan head should be correctly aligned by the Bio-Rad technical personnel during installation of the instrument, the scan head should be checked periodically to make sure there has been no movement between the scan head and the microscope. If the scan head is moved to another microseope, re-alignment will be essential. Aligning the scan head is a reasonably simple operation. The Scan Head should be positioned in approximately the correct position (deterrnined by the length of the connecting tube). The microscope and scan head should be connected with the enclosed connecting tube. There are two alignment procedures required; the first is to align the laser scan centrally, and the second is to focus the scan onto the back focal plane of the objective. To align the laser scan centrally, first make sure the plastic prism "bulls eye" provided by Bio-Rad is screwed into one of the objective sockets on the microscope. While the laser is scanning, gently move the scan head to align the scan to the centre of the "bulls eye". Once the scan is central, and while continuing to scan, gently turn the threaded turret on the scan head (microscope-coupling tube) until the scanning spot in the "bulls eye" stops flickering (pulsing). 1fthis cannot be achieved, try tuming the turret in the other direction. The laser should now be a spot of light in the centre of the "bulls eye" with only a hint of "flickering" as the laser scans. Once the scan has been aligned, both the microscope and the scan head should be bolted tightly to the table to prevent accidental movement. The sc an head should not need re-aligning into the microscope again unless either the microscope or scan head is moved. Low/High power on a Kr/Ar laser For cntlcal appllcabons (such as real coIour transmisslOll lmag ng) sWltch the laser 10 high power. The sWltch Is Iocated on the side of the laser. Don't forget - switch back to low powerl Appendix 1: Confocal Microseopes Bio-Rad 369 Optical Filter Blocks The Bio-Rad MRC-1024 scan head contains two removable optical filter blocks that house both dichroic mirrors and sometimes, suitable Don't forget - barrier filters (further barrier filters are provided on computer you must physically change the controlled rotating wheels in front of each PMT tube within the scan filter blocks In the scan headl head). Several different optical filter blocks are available and can be used in various combinations for different labelling requirements. Below is abrief description of the characteristics of the filter blocks available for the Bio-Rad MRC-l 024 scan head, with the most common filter blocks described in more detail shortly (Figure A-7, Figure A-8 and Figure A-9). Optical filter block combinations used for specific imaging methods are also described in some detail (Figure A-IO, Figure A-ll, Figure A-12). A requested change of filter block on the menu screen of the LaserSharp software requires a physical change of filter block within the scan head. Changing the filter block is a simple process of depressing the button on the top side of the filter block to release the catch and lifting the filter block out of the scan head. The replacement filter block is then simply pressed into place in the sc an head such that the catch "clicks" to indicate the filter block is correctly seated. Filter Blocks for the Bio-Rad MRC-1024 Confocal Microscope Krypton-argon-ion laser Tl: (tripIe dichroic rnirror) for use with the krypton-argon-ion laser - rellects 488, 568 and 647 nrn. T2A: (560 DRLP) dichroic rnirror for splitting the green (PMT2) and red (PMTI) Iluorescent light. Live cell imaging with any laser BI: bearnsplitter optical filter block where 80% of all light is transrnitted and 20% rellected. Used in position I for live cell studies, and in cornbination with the Tl tripIe dichroie mirror in position 2 for backscatter imaging. Argon-ion laser Al: (527 DRLP) for use with the argon-ion laser for duallabelling - rellects 488 and 514 nrn. A2: (565 DRLP) for splitting the green (PMT2) & red (PMTI) Iluorescent light. VHS: (510 DRLP) Rellects 457 & 488 nm blue light - for use with Ar-lasers for Iluorophores such as Lucifer yellow. Green ReNe laser EG I: rellects 488 & 543 nm for duallabelling with argon-ion/green HeNe lasers. E2: (560 DRLP ext R) splits Iluorescent light into greenlblue (PMT2) and red in PMTl. Specialised optical filter blocks OPEN: contains no filters or dichroic mirrors for single colour irnaging. SA2R: (610 DRSP) contains emission filters 580/32 and 640/40 for SNARF @ 514 nm excitation. SK2R: (605 DRSP) contains emission filters 570/40 and 640/40 for SNARF @488 nm excitation. FF2R: contains emission filters 600LP and 530/40 for Fura red plus Fluo-3 imaging. UV laser UBHS: rellects 351, 363 and 488 nrn light. INI I: (380 DCLP) rellects UV lines. INI 2: (440 DCLP) splits violet (for example, Indo-l) into PMT2 and blue light into PMTl E2: (560 DRLP) splits green and blue light into PMT2 and red light into PMTl. Note: DRLP = Dichroic Long Pass Filter & DRSP = Dichroic Short Pass Filter Appendix 1: Confocal Microseopes 370 Bio-Rad T1 Filter Block High percentage cf wavelengths between laser lines passes through this mirror 100% Transmission All wavelengrhs, excepl for V9/)' narrow regions afOUfld 488. 568 aOO 647 nm. pass /JIrough thls mirror Se/ective reflectlon of bllIfI. ye/low. & red laser lines mirror Reflectance ~~~~~~~~~~~~-p~~~~~-100% 700 488 568 647 Blue laser em ission Yellow laser emission Red laser emission Wavelength (nm) Figure A-7. MRC Tl Optical Filter Block. The Tl optical filter block is designed to reflect each ofthe 488 nm (blue), 568 nm (yellow) and 647 nm (red) laser lines ofthe krypton• argon-ion mixed gas laser. Wavelengths outside these three regions can pass through the dichroic mirror. This optical filter block is used in the position closest to the laser intake when doing standard dual and tripie labelling (Figure A-IO. The Tl filter block can also be placed in the 2nd filter block position (away !Tom the laser input) when doing backscatter (reflectance) imaging (Figure A-12). T2A Filter Block 100% Ye/low & redlighl Transmlssion passeS Ihrough ... ths clichrolc 560 Long Pass dichroie mirror Jf Se/ectlve reflectlon Dichroic 01 b1ue & gffHNI lig mirror ~ ~ RefIecIance 100% 1500 T 600 r 700 .... _-- &<7 - Wavelength (nm)--- Figure A-8. MRC T2A Optical Filter Block. The T2A filter block is designed to split red and green wavelengths of light. Short wavelengths (blue and green) are efficiently reflected by the dichroic mirror, whereas longer wavelengths (yellow to far-red) readily pass through the mirror. This filter block is used in the 2"d position (away from the laser intake) in combination with the Tl tripie dichroic mirror (in the first position, near the laser intake) for dual and tripie labelling. Appendix I: Confocal Microscopes Bio-Rad 371 B 1 Filter Block 80% of sM wavelengths PlJSS throug/l 'his mlrror 20% 01 1_ lighl reftected lowards Ihe sam.pIe JI 20% of 8' W8Ve/8nglhS Dichroic 81'81'8f1fH:.1ad mirror Reflectanoe ~~~~~~~~~~~~~~~-100% 500 600 700 4U 564 807 ------Wavelength (nm)--- Figure A-9. MRC BI Optical Filter Block. The B I filter bloek is designed to refleet 20% of all wavelengths of light, and to transmit the remaining 80% of all wavelengths. The filter bloek is partieularly suitable for live eell imaging where maximum sensitivity of fluoreseenee deteetion is required (Figure A-Il). The B I filter bloek ean also be used in eombination with the Tl tripie diehroie mirror for baekseatter (refleetanee) imaging (Figure A-12). Open Filter Block, contains no dichroic mirrors or filters, resulting in all light being directed to PMTl. Placed in the position in the scan head furthest away from the microscope. Don't forget that when "OPEN BLOCK" is selected on the computer the Open Block must be physically inserted in the scan head. 640SP (Short Pass) Permanently installed dichroic mirror (in 3-PMT systems) for directingfar-red light into PMT3. For s~cilllillbd/ing Ilppliclllions iI is po ib/~ to ;nstlllJ yo"r OK'n diclrroic ",i"on tlnd filters. Appendix I: Confoeal Mieroseopes 372 Bio-Rad Emission Filter Wheels Within the MRC-1024 scan head are emission filter wheels that are controlled by the LaserSharp software. Emission Filters These optical filters are used to prevent unwanted When using the krypton-argon-ion laser: excitation laser light entering the PMT tube, and to more closely define the region of the light spectrum that will PMT1: OPEN, 585LP, 605/32. OG515,680/32 be detected by each channel on the confocal microscope PMT2: OPEN, 522/35. Blue Reflection. (for dual and tripie labelling applications). The PMT3: OPEN. 680/32 combination of optical filters available will be dependent on the lasers you have installed on your When using the argon-lon laser: system. PMT1: OPEN OG515. 580/32. 585LP.680/32 Photomultiplier Tubes (PMT) PMT2: OPEN. 540/30. Blue Reflection The photomultiplier tubes are very sensItIve light PMT3: OPEN. 680/32 detectors that are mounted within the scan head. The MRC-I024 contains three separate photomultiplier tubes, which allows simultaneous 3-channel fluorescence imaging. There is aseparate "pinhole" (confocal iris) and an individual excitation filter wheel associated with each PMT. Controller Box The controller box for the MRC-I024 houses the electronic components for operating the confocal microscope. The controller box houses the "frame grabber" card, which is where the image is initially collected before transfer to the computer. The transmission detectors are located within the controller box, with a fibreoptic cable transferring the transmitted light from the microscope down to the detectors. The controller box should be tumed on fully (not just "stand-by" mode) before the computer is tumed on. If the computer attempts to complete the initialisation routine without the controller box being tumed on, an error will occur when the computer attempts to down-load control programs to components within the controller box. The controller box should be tumed to "stand-by" mode when not in use. Computer Requirements The MRC-1024 is usually installed with an IBM compatible DELL computer, although earlier versions of this microscope were operated with a Compaq computer. The more computer memory (RAM) and the faster the video card then the faster the 3D reconstruction will be and the smoother the "rocking" motion when displaying 3D images. Neither a high-end computer, nor a large amount of computer memory is required for the actual collection of confocal microscope images. A large hard disk is essential for collecting confocal microscope images. This is particularly important when collecting optical slices for 3D reconstruction or aseries of time-Iapse images. Imaging on a confocal microscope can very quickly result in the collection of a very large amount of image data. Regular removal of the collected images to a more permanent form of storage, such as a CD, is most important. The software for operating the MRC- 1024 confocal microscope was originally written for OS/2, an operating system that is now obsolete. A Windows NT version of LaserSharp is available for operating the MRC-I 024 confocal microscope, but obtain help from Bio• Rad or other users before upgrading so as to avoid the many problems that can be caused. People who have upgraded successfully are convinced the effort is worthwhile as you then have "in house" support for a Windows operating environment, which greatly facilitates such things as linking to your network and installing additional drives etc. Hardware set up for specific imaging methods Thcre are a number of "standard" optical filter block combinations that are routinely used for a variety of fluorophores. These are described in some detail on the following pages. Appendix 1: Confocal Microseopes Bio-Rad 373 Dual and Tripie labelling - T1 and T2A Filter Blocks T1 • Tripie Dichroic 100% 01 nuorescenoe between la_Iones doreded ID PMTs Il '*' '*' (Position 1 -cJose 10 laser) The T1 (tripie dichroie) reflects all three laser lines from the krypton-argon-ion laser lines towards the sampie. and allows 100% of all fluorescence wavelengths between these laser lines _100"A..... d-.ctl - to be directed to the PMTs. -- T2A • 560 DRLP (Position 2 - away from laser) .... The T2A (560 LP dichroie) reflects -_ Wavelength (nm)--- shorter wavelengths (green) into PMT2 --- and allows longer wavelength (red & far red) light to be transmitted. ~IOPMT' ~------~~====-==------~··100 % ...... *Iwok: --..., ...... 00II _ It. ~ -- _.It.&tOSP ___...... ,IIgI'II_ T...... ",..soon PIoIT3 __...:IIIgI'IIIO_PMT 1 --- Wavelength--- (nm) 560 lP dlehtole - a mirror that trilosmlts wavelengths Iooger (Loog Pass) thao 560 nm. 640 SP diven. - a morror that traosmits waveleogt/ls s/lof18(ShortP $)than640 nm. PMT1 =Red channel PMT2 =Green channel Microscope PMT3 =Far-red channel Figure A-lO. MRC Tl and T2A Filter Block Combination. Tbe TI and T2A optical filter block combination is ideal for dual and tripie labelling using fluorophores such as FITC, Texas Red and Cy5. The TI block directs all laser lines (blue, yellow and red) to the sampie, and the fluorescent green, red and far-red light is transmitted to the photomultiplier tubes. Tbe T2A block contains the 560LP dichroie mirror, which splits the emission into green and longer (red) wavelengths. The 640SP diverter (perrnanently installed in 3-PMT systems) results in far-red light being directed into PMT3. Appendix 1: Confocal Microseopes 374 Bio-Rad Live Cell Work - B1 and OPEN BLOCK Filter Blocks B1- Beam Splitter 80% 01 all wavelenglhs direcled 10 PMTs (Position 1 - c/ose 10 laser) The 81 filter block renects 20% 01 all JI' laser light towards the sampie, and allows 80% 01 all fluorescence wavelengths 10 be directed 10 Ihe PMTs. OPEN BLOCK (Position 2 - away 'rom laser) .... -_- ~ ---Wavelength (nm)--- The absence 01 any dichroie mirror or lilter in position !wo results in all '- . nuorescenl and renected light being ~ ,...-______..., directed into PMT 1. All f1uorescence directed 10 PMT1 +100% c: o TrIIt'I$mi$$ion 'in 1/1 'E CI) c:B CI) (,,) 1/1 eo :::l Li: ---- Wavelength--- (nm) PMT1 = Green. Red and Far-red PMT2 & PMT3 are not used microscope Figure A-IL MRC BI and Open Block Combination. The Bland OPEN filter block combination is ideal for live cell work as 20% of all laser light is directed to the sampie, and all fluorescent light is directed into PMTI with approximately 80% efficiency (longer wavelength red light is directed to PMT3 by the 640SP diverter). This results in less laser light reaching the sampie, but excellent efficiency in the collection of fluorescent light. Appendix I: eonfocal Microscopes Bio-Rad 375 Back Scatter Imaging - B1 and T1 Filter Blocks B 1 • Beam Splitter ~ 80% of all wavelengths directed 10 PMTs c (Po.ltJon 1 - cIose to /8ser) o '(ij JI The 81 flHer block reftects 20% of all CI! three laser lines lowards the sampla, 'E ID and allows 80% of all lluorescenl Of lmIo GI _ IUIII backscattered (reflected) light to be 8c: directed 10 the PMTs...... ID u CI! -• o~ ;:, Li:: T1 - Tripie Dichroic (Po.ltJon 2 - awey from laser) .., The T1 (tripla dichroic) filter block when .. placed in position 2 reftects all three --- Wavelength (nm)--- backscattered krypton-argon-ion laser lines 10 PMT3, and anows 100% of all lIuorescence wavelengths between the laser lines 10 be directed to PMT1 and PMT3. T...... tion ,. .. , Wavelength--- (nm)--- PMT1 =Green & red fluorescence PMT2 =Reflected blue light PMT3 = Far-red fluorescence Figure A-12. MRC Back Scatter (Reßectance) Imaging. Backscatter imaging, also known as reflectance imaging, is a highly sensitive method of imaging, which gives excellent resolution. Some biological material is naturally highly reflective, or specific structures can be imaged by using silver enhanced gold immunolabelling. In this filter block combination all backscattered (reflected) light is directed to PMT2, and all fluorescent light is directed to either PMTl (red and green light) or PMT 3 (far-red light). Appendix I: Confocal Microseopes 376 Bio-Rad TROUBLE SHOOTING (MRC AND RADlANCE) Laser out of alignment See page 6-11 ofthe MRC-1024 manual. Warning - do not attempt to align the laser unless you have been instructed how to do so. Scan head not correctly MRC: Adjust mirror MI (see Figure A-6 on page 367). Take care to make only small aligned into microscope adjustments while aligning the laser within the "bulls cye" of the prism on the microscope. Radiance: Refer to section 7.2.2 of the Micro Radiance operating manual. Two adjustments can be made within the "neck" of the sean head where the sc an head attaches to the microseope. One adjusts the lateral alignment of the laser; the other adjusts the focus of the laser. Laser not correctly MRC: Adjust mirror M4 to align laser into PMTl (the red channel when using the aligned into PMT tubes Tl/T2A filter block combination for dual/triple labelling). Adjust mirror M5 to align mirror into PMT2 (the green channel when dual/triple labelling). Radiance: Alignment into the PMT tubes (now houscd in the controller box) is factory set and should not require adjustment by the user. L_~of~ Laser alignment See "diminished sensitivity" above. Objective not planer An objective that does not have a completely flat field of view (a Planar lens) will result in dark areas of the image towards the edges. This problem is often more obvious on a low magnification lens. A lower quality lens will also display considerable unevenness in the field of view. Considerable improvement in the lack of planar field of view ean often be accomplished by re-scanning the image at a higher zoom. 0"'" d...t Lossof647nmredline Check to see if the red line is present by setting the scan to 647 nm line only (using (Kr/Ar laser) LaserSharp) and looking at the sampIe (from the side, not through the eyepieees of the microseope) while imaging. First wait for a further 15 minutes to make sure the laser is fully warmed up. Dark screen: Pickup Swing the bar on the transmission mirror towards the back of the microseope. The mirror incorrectly set transmission mirror is located above the condenser on an inverted microseope. The mirror position should be automatically set by the software when attempting to change the gain on the transmission setting when using the Radiance series of confocal microscopes. Bright screen: set gain Use the SETCOL LUT to set the gain and black (offset) levels to their optimum and offset controls. settings. SoftwIrt probImII Software frequently Make sure that there is sufficient room on the hard disk for the system swap file (try ---~~-- "freczes" to have at least 500 MB free). I'oorqDlllll} ..... Immersion oil on a dry Clean objective with ethanol and lens tissue to remove any residual oil that may have L-___~ lens accidentally stuck to the lens. Incorrect setting on lens Some lenses have a correction collar for oil, glycerol or water immersion, make sure that the eorrect setting is selected for the immersion media in use. J...,.t~ _ ~~I0~"'~ Vibration during scan Mount the microscope and scan head on a vibration-damping platforrn. Placing the scan head and microscope on a metal plate that is placed on the top of two small bike tubes can create a simple vibration platforrn. The air cushion created will damp out most vibration. llluntllaof ..... twIIm Vibration during scan Vibration may not be obvious with a single scan, but may manifest itself as a blurred .... K*-CGIrIcdoa image after averaging several scans when using thc KaIman collection mode. 00 not test suspected vibration problems using live cells as slight movement of the cells during imaging can cause "blurring" effects. Cell movement when Try "SLOW" scan or line averaging instead of "KaIman" screen averaging for imaging lives cells improving image quality. Interfering electrical Electrical equipment, particularly computer screens can cause banding patterns on the equipment collected image. Placc any additional equipment or screens further away from sensitive elcctrical components. Laser unstable Have laser serviced by service engineer. Press "Ctrl-L", or run the lasercheck script on Radiance systems, to obtain a listing of the anode current, laser power and hours used for the laser. This information will be useful for a suitable technician to diagnose the problem. Appendix 1: Confocal Microseopes Bio-Rad 377 BIO-RAD LASERSHARP SOFTWARE The latest version of LaserSharp software produced by Bio-Rad (LaserSharp2000, LS2K) is designed to run under Windows NT/2000 (currently LaserSharp A nalysis used for the Radiance 2000/2100 and Micro The analysis component of the LaserSharp program Radiance confocal microscopes). A Windows NT can be loaded onto other computers for opening Bio• version of LaserSharp is also available for the Rad ·Ple- files with all microscope information intact MRC-I024 confocal microscope. The spectral imaging Radiance 2100 Rainbow confocal microscope is operated with the Bio-Rad SpectraSharp software. The original software used for controlling the MRC-I024 confocal microscope was called LaserSharp, and was developed to run under the OS/2 operating system. The OS/2 operating system was an operating system developed by IBM but is no longer being produced or supported. OS/2 did have significant advantages over the earlier versions of Windows and the DOS operating system. This was particularly important in the way the OS/2 operating system easily handled very large file sizes. However, with the advent of more advanced vers ions of the Windows operating system the OS/2 operating system is no longer advantageous. In fact there is now often considerable difficulty obtaining expert advice on the OS/2 operating system. If you have an older Bio-Rad MRC confocal microscope using the older OS/2 software you should contact Bio-Rad about upgrading to the latest Windows version. An emulation / data processing version of LaserSharp2000 is available for people still using an OS/2 based instrument. This software can be used to manipulate the "PIC" format files and give you some idea of the format of the new LaserSharp interface. The LaserSharp software is divided into two components - acquisition and analysis. The acquisition component can only be run on the confocal microscope computer and is used to collect the images from the microscope and to control the scan head settings. The analysis part ofLaserSharp can be used on the confocal microscope computer, or loaded onto another computer, which can free up valuable time on the confocal microscope. Bio-Rad has also developed aseparate pro gram called LaserPix, which is designed as a "stand alone" image analysis program for confocal microscope images. This program is based on the popular image analysis pro gram "ImageProPlus" with added features for confocal microscopy. LaserPix will run under Windows NT/2000 and can be loaded on a separate computer to the one attached to the confocal microscope. LaserPix does require aseparate license and associated "dongle" before installation can proceed. The following information is intended to give you an introductory overview of the LaserSharp software. If you do require more detailed information please refer to the Bio-Rad user manual, or probably a much better way - ask someone who is already using LaserSharp! The proprietary Bio-Rad "PIC" file format can be read by using a Photoshop plugin (available for download from Bio-Rad, fto: //fto.genetics.bio-rad.comlPublic/confocal/). Confocal Assistant, a small free imaging processing pro gram designed to handle Bio-Rad "PIC" format images, is also available from the Bio-Rad FTP site. LaserSharp Login Procedure LaserSharp software set up for a multi-user system should be installed with a password for each user to log on to the instrument. This password is not a security system, but simply a means of directing all of your collected images to your own subdirectory on the computer's hard diskoAnyone who has logged on under their own password has full access to your files! If you use the LaserSharp Iogin procedure you will also have in your subdirectory a copy of all your "methods" and colour Look Up Tables (LUT) . These files are copied from the ·Default" user directory when you are registered as a new user. "Methods· that you save will be retained in your own subdirectories. "Methods" developed by other users of the microscope will not be directly available to you - unless they are copied into your subdirectory. The Login password is NOT a security systeml Appendix 1: Confocal Microseopes 378 Bio-Rad Image Collection Panel The Bio-Rad LaserSharp software can be operated using a single screen, in which case the control panel and image being collected will be displayed as shown in Figure A-13. Altematively, a double computer screen can be used with one monitor displaying the main menu and associated control panels and the other screen displaying the images as they are collected. The images displayed on the Bio-Rad display screen are the output of one of 3 "mixers", rather than the output of individual collection channels. In many applications the output for mixer 1 would be simply 100% PMTl and the output for mixer 2, 100% PMT2. However, one can readily mix different combinations of the channels. For example, when multi-channel labelling you can subtract, say, 10% of PMT2 from PMTl by making Mixer 1 a combination of 100% PMT 2 and -10% of PMT 1. This is a simple and dynamic method of eliminating unwanted bleed through in multi-labelling applications. Maln eontrol menu and eontrol leon. Figure A-13. Bio-Rad LaserSharp Main Display Screen The Bio-Rad LaserSharp software can be used on a single computer monitor, in which case the control panel and collected images would be displayed as shown above. However, the software does support the use oftwo monitors, where the control panel and other menu options can be displayed on one monitor and the collected images displayed on a second monitor. On-screen image display is the output of individual "mixers" as described above, and is not necessarily the output from a single detection channel. Appendix I: Confocal M icroscopes Bio-Rad 379 Main Control Panel The main control panel (Figure A-14) is the central part ofthe LaserSharp software for controlling the Bio-Rad confocal microscopes, and displays in one simple panel all ofthe main control fimctions you will need to operate the instrument. Scanning is initiated by pressing the "Start scanning" button or simply depressing "F12" on the keyboard. Diagram of optIc.lllayout for adjustment of filters. dichroics elc see Figure A-15 Start scanning (er press -F1T) Move around the field of view (only worXs when zoomed) Sesn position in field of view Selection of scan speed mode ~~""""'-::11.... t- Selection of box size in pixels ~ at _,.-. ~ IQ x4) _.r.~--:-~ Don'I forgel to seled Ihe COfTed objectlve - olherwise the scale bar. pixel slze elc will be incorrect, Only a single cha.nnel (denoled "~) is lumed on in Ihis "melhod" SeI iris (confocal pinhole) to orte AJry dlsk Adjusl gain controI (in some instruments you directly adjust the Pf.AT voltage) c: 01 Focus motor control for coIlec1ing aseries { .100 of optical slices Figure A-14, Bio-Rad LaserSharp Main Control Panel The Bio-Rad LaserSharp main control panel is the central window through which aB of the other microscope controls are accessed. Pre-saved "methods" can be used to set up the controls on the instrument. Y ou can also save your own instrument settings fOT later recalL Appendix I: Confocal Microscopes 380 Bio-Rad Optics Control Panel The "Optic" control panel (see Figure A-15) displays graphically the optical settings for the confocal microseope. The settings shown in this diagram are "pre-set" when you select a specific "method". However, check these settings even when using a known "method" as one can quite easily inadvertently alter a method and still save the method under the same name. In fact, in multi-user facilities take great care in regard to filter settings when using specific methods if you don 't have your own login and personal file and method allocations. In this example the 405 nm violet laser diode (also known as asolid state violet laser) and the 543 nm green HeNe lasers are active. A long pass dichroic mirror (560DCLP) is used to split the fluorescent light into red (wavelengths longer than 560 nm) and green (wavelengths shorter than 560 nm). Various "blocking" and "emission" filters are used to further define the wavelengths allowed to enter each detection channel (only two, PMT2 and PMT3, are active in this example). The opticallayout shown in the "Optic" panel is a combination of components located in both the scan head and the controller box. " L ers Green HeNe laser and blue laser diode are active In this particular set up only two channels are active Figure A-15. Bio-Rad LaserSharp Optics Control Panel Selection of the "Optic" control panel allows one to change the optical filters and dichroic mirrors using a graphie display of the optics layout of the instrument. In this example the blne laser diode (13% power) and the green HeNe laser (14.6% power) are activatcd. The "red" (PMT3) and "green" (PMT2) image collection channels are active. Appendix I: Confocal Microseopes Bio-Rad 381 Mixer Control Panel The LaserSharp software has the unique characteristic that each of the collected images is created by the output of various detectors as determined by the user (or method used). Common practice is to assign 100% PMT I to mixer one, as shown below. However, other combinations of detectors can be assigned to mixer 1. For example, you could assign a level of minus 10% of PMT 2 to mixer one (in addition to assigning 100% of PMT I to mixer I) to correct for bleed through when dual channellabelling. This ability to display the output as a combination of different detection channels is a powernd tool for real-time dynamic bleed-through correction. MIXer 1 settJngs Only the "red" PMT channel is operating in this example Fignre A-16. Bio-Rad LaserSharp Mixer Control Panel Each of the image display panels in the LaserSharp software is the output of various PMT tubes or transmission detectors (calIed Mixers). In the above example Mixer I is comprised of 100% PMT2, without any input from any other charme!. However, one can assign set percentages of other detectors to any of the mixers, including subtracting apercentage of one PMT output from another PMT output (used in dual and tripie labelling applications to subtract bleed-through). Press "F12" to start scanning Appendix I: Confocal Microscopes 382 Carl Zeiss earl Zeiss Microscopy he earl Zeiss optics company has been producing superb optical instruments since the very beginning of the T commercial development of optical instruments. The coming together of the earl Zeiss lens manufacturing with the development of specialised optical glass from Qtto Schott, and the theoretical input on optical design from Ernst Abbe resulted in a highly innovative company dedicated to high quality optics. Although the cornpany has had a traurnatic history, including the splitting ofthe company into an East and West German entity - today the company once again operates as a single unit. The production and development of the laser scanning confocal microscopes is located in Jena, the town where earl Zeiss made his first microscopes. ZEISS CONFOCAL MICROSCOPES The latest laser-scanning instrument from earl Zeiss is the LSM 510 META confocal microscope (Figure A-17). This instrument is capable of multiple channel fluorescence imaging using both conventional beam splitting optics and the innovative spectral imaging META system where multiple overlapping fluorophores can be imaged simultaneously. Figure A-17. Zeiss LSM 510 META Confocal Microscope. The Zeiss LSM 51 0 META eonfoeal mieroseope is designed for multi-ehannel fluorescence imaging in the biological seienees. The META scan head and assoeiated optics is shown here attaehed to a Zeiss inverted research grade light microscope. The lasers, and the computer used to control the instrument are located under the microscope bench. This photo graph was kindly provided by Gavin Symonds, Carl Zeiss, Australia. earl Zeiss Web· httn'.,. fiv.,'Voi'\V zeiss.. deilsm, . or www zeiss. .. comlmicro Address: Carl Zeiss, Mikroskopie Carl Zeiss Micro imaging, Inc. Carl Zeiss Pty. Ud. 0-07740 Jena One Zeiss Drive 114 Pynnont Bridge Road Germany Thomwood, NY 10594 Camperdown, NSW 2050 USA Australia Phonc: +493641641616 + I 800 233 2343 1800112401 FAX: +493641643144 +1 (914) 6817446 +61 (02) 9519-5642 Appendix I: Confocal Microscopes earl Zeiss 383 Zeiss Confocal Microscopes Laser Scanning Confocal Microscopy Carl Zeiss has been producing microscopes since the Zeiss has continued 10 further develop and automate their conjocal very beginning oflight microscopy in the mid-1840's. microscopes, with a number of innovative derelopmenls in recent years, including the development of Ihe META - multi-array By the early 1900's Zeiss was a major manufacturer detector for spectral imaging. of high quality compound microscopes for both r-L-S-M-5-1-0-M-E-T-A--'-F-u-ll-y-co-m-p-u-te-r-c-on-tr-o-I-Ie-d-c-on-r.-oc-a-I' industry and the research community, microscope with both "conventional" The company was split into two at the end of WWII, detection channels and the multi-PMT with West German Zeiss being established in detector array (META channel). allowing the simultaneous detection of Oberkochen (near Stuttgart, West Germany) with the several fluorescent labels by analysing transfer of a large number of technical and scientific the fluorescence emission spectrum at staff from the Jena factory by the USA occupying each pixel within the image. forces. Zeiss continued in Jena as astate enterprise LSM 510 A fully computer controlled confocal (VEB Carl Zeiss JENA) of the German Democratic microscope with up to 4 fluorescence Republic. On re-unification of Germany, Zeiss was detection channels. again brought back together, with the production of LSM 5 PASCAL Fully computer controlled "personal" microscopes continuing In both towns, but the confocal mieroseope available in a research and development and continued innovative variety 01' eonfigurations, including a ehoice of lasers and detection ehannels. changes to the confocal microscope being established in Jena. LSM 510NLO Multi-photon version of the above LSM 510 and LSM 510 META. Fully Zeiss first commercially produced a laser scanning LSM 510 META automated microscopes, with a mirror confocal microscope (LSM 44) in 1982. This was connected Ti-Sapphire laser providing upgraded with the LSM 10 in 1988, The early Carl NLO ultra short pulsed infrared light for Zeiss confocal microscopes were designed for use in multi-photon rnicroscopy. L-.______-"--_-'- ____-'-'-- ___---' materials science (particularly microchip Earlier Zeiss Confocal Microscopes manufacturing) and were not sufficiently sensitive to The earlier Zeiss confocal microscopes were high quality be of general use in biology, With the introduction of instruments with superh resolution, hut with fewer detection the windows controlled 2-channel LSM 310 (1991) channels, a lower degree of automation and somewhat lacking in sensitivity, and therefore of limited use in the biological seiences. and 3-channel LSM 410 (1992) with individual pinholes, and greatly increased sensitivity, the Zeiss LSM 410/310 3- or 2-channel confocal microscopes, confocal microscope became highly regarded in the for use with inverted (410) or upright (310) microscope stands. Windows biological research community, control of scanning modes and The LSM 510 in 1997 has improved on the original advanced graphics boards with mega excellent design, particularly with the addition of pixel-resolution made these instruments suitable for both industry and biology. flexibility, speed and greater functionality of the software, This instrument is a highly versatile and LSM 10/44 2- or I-channel confocal microscopes. Built in the pioneering 1980's, these very sensitive multi channel confocal microscope, confocal microseopes were primarily The LSM 510 METAis capable of spectral imaging L-______---'-_d_e_si_gn_e_d_D_or_u_s_e_in_i_n_du_s_try_. ___--' using a multi-PMT array, This innovative design Fluorescence Correlation Spectroscopy (FCS) allows the simultaneous imaging of several Fluorescence correlation spectroscopy is used 10 sll/dy molecular fluorophores without the necessity for optical filters interactions by following random molecl/lar movement within a etc to collect specific "windows" of light. Several ,-d._efi_ln_e_d_m_ic_'_·o_sc_o_PI_·c_v_ol_u,m_e.______-., fluorophores can be separated into different ConfoCor 2 An FCS mieroseope tor use in the "channels" by automated analysis of the spectrum biological scienees. This instrument obtained. As many as 6 fluoro chromes can be readily can be combined with a variety of eonfigurations of the Zeiss LSM 510 separated In this manner, in addition to two and LSM 510 META eonfocal "conventional" single PMT detection channels, microscopes. Appendix I: Confocal Microscopes 384 Carl Zeiss Carl Zeiss also produces a high-quality but less versatile instrument cal1ed the LSM 5 PASCAL confocal microseope. This instrument is of similar design to the Zeiss 510 META, but without the META multi-PMT array channel. The LSM 5 PASCAL is significantly less expensive compared to the LSM 51 0 META confocal microscope, being designed for use in individuallaboratories as a "personal" confocal microseope, rather than being based in large University or Research Institute facilities. The ConfoCor2 fluorescence correlation spectroscopy instrument is a specialised addition to the conventional confocal microscope for studies on molecular interactions and movement of macromolecules. ZEISS LSM 510 META CONFOCAL MICROSCOPE The Zeiss LSM 51 0 META confocal microscope, as mentioned above, is a conventional confocal microscope based on the use of dichroic mirrors and optical filters with an additional META spectral imaging channel. The META channel can be used as a multiple channel imaging device, but the real power of the META channel is the ability to separate the fluorescence emission from several very similar fluorochromes by a process cal1ed linear unmixing - al10wing one to perforrn multiple labelling experiments using remarkably similar probes. A wide range of visible light and UV lasers can be connected to the Zeiss instruments. Modified vers ions of the LSM 510 META confocal microscope are also available for use with pulsed infra-red lasers for multi-photon imaging. Fibre optic connection for lasers Vibration isolation segment oftable Inverted light microscope Figure A-18. Zeiss LSM 510 META Seall Head. The LSM 510 META confocal microscope sc an head is shown attached to a Zeiss research grade full y automated inverted microscope. The sean head eontains the seanning mirrors, diehroic mirrors. optieal filters, detec tors (inciuding the META 32-PMT array detector) and the confocal pinholes. The lasers are located remote from the scan head, \Vith the laser light being directed into the sean hcad via fibreoptie connections. In this photograph the microscope and att"ched confocal microscope sean head are shown mounted on a vibration isolation segment of the main instrument work dcsk. This photograph was taken at the Baker Heart Research Institute, Melbourne, Australia. Appendix I: Confocal Microscopes Carl Zeiss 385 Components 0' the Zeiss 510 META The Zeiss LSM 510 META laser scanning confocal microscope can be eonfigured for visible light, UV light or multi-photon microseopy on either upright or inverted microseopes. DaIp The Zeiss LSM 510 META instrument is based on both a multi-ehannel META detector and two dichroie mirror / optieal filter based channels. A very high-end eonfoeal mieroscope that has great versatility, but due to the relatively high eost is often loeated in central facilities rather than individuallaboratories. Sela Had The Zeiss LSM 51 0 META sean head is mounted direetly onto a Zeiss research grade inverted or upright microseope. The sean head houses all of the opties, pinholes and deteetors neeessary for confoeal imaging. The sean head can be moved from inverted to upright mieroseope if required. META deteetion ehannel: The META deteetion ehannel eonsists of a speetral separation diffraetion grating and a 32-PMT array that ean be used as additional channeIs for fluoreseenee imaging, as a multi• channel detector - or as a speetral imaging channel for multi-eolour labelling. One of the unique advantages of the META deteetor is that a computer algorithm called "linear unmixing" can be used to separate fluorophores with very similar emission speetra. Conventional detection ehannels: Two "conventional" deteetion channeIs that employ dichroie mirrors and optieal filters to separate out different wavelengths of fluorescent light. Independent photomultiplier tubes for each channel are used to detect the fluoreseent light. Diehroic mirrors: Computer seleeted dichroie mirrors to both separate the fluoreseence emission from the excitation light (the primary dichroic mirror for conventional and META channels) and to further separate the fluorescent light into various regions ofthe light speetrum (only used on the conventional ehannels). Barrier filters: A computer-controlled wheel of removable barrier filters is mounted in front of each of the conventional photomultiplier tubes. The META deteetion ehannel does not require barrier filters as the light is separated into the light spectrum by a diffraction grating. Variable size eonfoeal iris: A computer-controlled variable size "pinhole" is mounted in front of each detector channel, including a single variable size confocal iris for the META channel. For optimal resolution and sensitivity, the META pinhole can be aligned in x, y & z and the other pinholes in x & y. Light deteetors: Individual photomultiplier tubes are used for each of the "conventional" detection channels, and an additional 32-PMT array is used for the META deteetor. Up to 4 optional external non• descanned PMT detectors are available for detection of fluorescence generated by multi-photon excitation. Seanning mirrors: X & Y fast galvo scan mirrors with zoom, bi-directional & scan rotation capabilities. '--' Lasers: A wide range of lasers can be attached via fibre optic conneetion, including the Kr/Ar, Ar-ion, '----"= HeNe, 405 nm violet diode laser and UV-Ar-ion lasers. Ti:sapphire multi-photon lasers ean be attached to suitably eonfigured instruments via fibre optic connection or direct coupling. Line seleetion and laser light intensity attenuation: Laser Iines are selected and may be rapidly switched using aeousto-optieal tuneable filters (AOTF) under software eontrol. AOTF filters also eontrol laser intensity (giving accurate levels ofbetween 0 and 100% ofthe eurrent laser power setting). ~ The sean head is attached to either an upright or inverted Zeiss automated research microseope. High quality Zeiss objectives are most important for producing a high-resolution confocal image. The seanning laser light enters the mieroscope just below the objective in an inverted microseope, resulting in an image quality at least as good as that on an upright microseope. Multi-ehannel fluoreseence, backseatter, transmission. Image eolleetion box sizes user selectable from I x 4 up to 2048 x 2048 pixels. Seanning modes include spot, line, free-hand line, ROI, z-stack, zoom, panning and rota ted scanning. These may be combined with time lapse imaging and spectrally resolved imaging (Lambda Stacks). Simultaneous and sequential multi-channel scanning optionally combined with localized bleaehing. Online calculations such as emission ratio and linear unmixing (Online Fingerprinting). 12-bit data acquisition and processing, saved as 8-bit or 12-bit image files. Single channel transmission detection using a photo multiplier detector. DlC, Phase and VAREL images can be obtained using the transmission detection ehannel and suitable microscope optics. The instrument is fully computer controlled with no programmable manual control device. High-end Windows based computer used to control the confocal microscope and collect the images. Dual computer screens may be used to display the control panel and images on separate monitors. The Zeiss proprietary software for controlling the microseope, image colleetion, 2D to 4D image manipulation, 3D- and 4D reeonstruction, visualization, animation and image file management system is operated under Windows NT/2000. The software can be readily loaded onto other instruments not associated with the microscope (such as a laptop) and the basic image database management utilised to access both the images and the information on microscope settings that are stored with the images. LSM 510 META - with META and "conventional" channels. LSM 510 - various configurations with up to 4 single-channel PMT detectors. LSM 510 !liLO and LSM 510 META NLO Multi-photon - having both confocal and multi-photon capabilities; optionally equipped with up to 4 additional external PMT detectors. LSM 5 PASCAL - "personal" confocal microscope for single-user applications. Appendix I: Confocal Microseopes 386 earl Zeiss Zeiss LSM 510 META Scan Head The relatively large LSM 510 META sc an head (Figure A-18) contains all of the optical components required for scanning the laser beam across the sampie and separating the t1uorescence emission into various regions of the light spectrum. This includes the dichroic mirrors, optical filters, diffraction grating, META detector and photomultiplier tubes. The Zeiss LSM 510 scan head is directly connected to a conventional Zeiss research grade light microscope, both of which are mounted on a vibration isolation segment of the microscope work bench. The sc an head can be readily moved between various microscopes ifrequired. The scan head can be connected to either manually operated or fully automated microscopes. When the sc an head is connected to a flilly automated microseope, changing between bright-field imaging and confocal imaging is a simple matter of pressing a "button" on the Zeiss LSM controlling software. The scan head is fully motorised, allowing full computer contro!. The lasers used for irradiating the sampie are connected to the scan head via fibreoptic connections, except for the pulsed infrared laser used in multi-photon imaging, which is directly optically coupled to the scan head. A schematic diagram of the optical components of the Zeiss LSM 510 META scan head is shown in Figure A-19, with the on-screen diagram used for changing variolls scan head settings shown in Figure A-21. A number of different primary dichroic mirrors can be software selected (for example, the tripie dichroic mirror that reflects 488, 543 and 633 nm light as shown in Figure A-21) for the separation ofthe irradiating laser light from the fluorescent light emanating from the sampie, shown as optical filter wheel (I) in Figure A-19 and Figure A-21. A second removable mirror, shown as optical filter wheel (2) in Figure A-19 and Figure A-21 is used to direct the fluorescent light to channel 2 and 3 (Ch2 and Ch3). The mirror shown in position "2" in Figure A-21 can be replaced by a dichroic mirror to direct specified regions of the emission spectrum to either of these two channels. Further specified wavelengths can be directed to the META channel, denoted ChS in Figure A-19 and Figure A-21 by choice of the correct dichroic mirror at position "2" in Figure A-21. Alternatively, the mirror at position "2" can be removed altogether to allow all fluorescent light to be directed to the METAchanne!. A third dichroie mirror filter wheel, denoted optical filter wheel (3) in Figure A-19 and Figure A-21 is used to separate the emitted fluorescent light into different regions of the light spectrum for detection in either channel 2 (Ch2) or channel 3 (Ch3). The META channel utilises a diffraction grating, denoted (7) in Figure A-19, to separate the emitted fluorescent light into its constituent wavelengths, which are then detected using the 32-PMT array META detector (ChS in Figure A-19 and Figure A-21). There are independently controlled confocal pinholes for each of the "conventional" detection channels, but only one confocal pinhole for the META channel (Figure A-19). Alignment of the confocal pinholes associated with each of the single PMT detection channels can be adjusted in x and y using software control, and the pinhole use for the META detector can be adjusted in x, y and z directions. Pinhole alignment is important for gaining maximum sensitivity from each of the channels -- and should be perforrned each time the dichroic mirror/optical filter settings are changed. Lasers A wide variety of visible light lasers can be readily connected via fibreoptic coupling to the Zeiss 510 META scan head. This includes the commonly used lasers such as the argon-ion and krypton-argon-ion lasers and helium• neon lasers. A wide range of lasers and laser lines can then be software selected, including high-speed switching for line by line Multi Track imaging. UV lasers can also be connected by fibreoptic coupling to the scan head. These lasers are usually relatively high• powcred argon-ion lasers in which the shorter wavelength UV lines are selected. There is considerable difficulty (and danger) associated with using UV lasers for confocal microscopy. The relatively short wavelength solid-state lasers, such as the 405 nm violet laser diode, may eventually replace most uses ofthe UV laser. A pulsed infrared laser for multi-photon microscopy cau also be connected to the scan head. Zeiss has experimented with both fibre optical coupling and direct connection of the laser, although the simpler direct coupling of the laser is favoured for most installations. Appendix l: Contllcal Microseopes Carl Zeiss 387 PMT tubes for "conventional" detection channels Fibre optoc: OUtput 10 spedr8I anaIyIer Dlchroic mirror tor separation of fluorescence eml slon Main dichroic mirror for directing ~ the laser light to the sampie \ Scanning mirrors FigurP A-19. Zt>iss LSM 510 META Scan Head La)"out. The oplieal layoul inside Ihe leiss LSM 510 META scan head shows Ihe dichroie mirrors ( 1. 2 .nd 3) .nd oplical lihers (5 and 6) for conventional splining of Ihe fluorescent lighl (IWO ch.nnels. Ch2 ,and eh3. in Ihis ex.mple). as weil as a diffraclion grating (7) in Ihe 3" channello separale Ihe lighl, \\"ilh a META deleelor (ChS) Ihal uscs an array of 32 phOlomultiplier tubes 10 deleellhe fluores Appendix I: e onfocal Microseopes 388 earl Zeiss Single Track and Multi Track Image Collection Image acquisition can be perfonned using the single tack mode (shown in Figure A-2l), where a single sc an of the sampie is made using either single or multiple channel acquisition. One can also collect images in the "Multi Track" mode (the "Multi Track" tab is shown in the Configuration Control panel in Figure A-2l), where individual screens or lines are collected using specified eollection parameters. For example, when duallabelling using Cy2 and Cy3 it is possible to colleet both channels simultaneously using the "Single Track" mode. However, ifbleed through is a problem then each individual ehannel can be exeited separately and the fluoreseent light collected separately using the "Multi Track" mode. This ean be done by collecting, for example, the Cy2 fluorescenee using all neeessary settings for this dye (inciuding the optimal laser line for excitation), and then eollecting the subsequent image using settings (and appropriate laser line) for the Cy3 dye. Sequential frame collection is fine when collecting images from fixed material, but when imaging live cells any slight movement between collection frames will detract from accurate image registration when comparing areas of colocalisation. A much better way of sequentially collecting duallabelled sampies is to use the "Multi Track" mode using line collection. In this case each individual scan line is eolleeted using a different laser line, with only one ehannel active at each line scan. The resultant images will have minimal bleed through between individual collection channels. Conventional Detection Channels In addition to the META channel, described in detail below, the Zeiss LSM 51 0 META confocal microscope scan head also contains two conventional detection channels. Each conventional channel eontains a single highly sensitive photomultiplier tube that is designed to detect any light that is directed to it by means of dichroie mirrors and optieal filters. Each conventional detection ehannel has a confocal pinhole that can be aligned (in x and y) and varied in diameter. The gain and offset can also be adjusted independently for each photomultiplier tube. These single ehannel detectors can also be combined with the META channel to create a highly versatile instrument with a large number of imaging channels. META Channel The META ehannel consists of a diffraction grating, which is used to split the fluorescent light into its component colours, and a multi-PMT array (32 individual PMT tubes arranged in an array) to detect the light. The META ehannel can be used as up to eight "eonventional" channels (in addition to the single PMT channels already available on the instrument), or a method called "linear unmixing" can be employed to derive the contributions from the individual fluorophores by means of spectral infonnation. The META channel, when used as a multi-band pass optical filter (Figure A-25), has on-screen sliders to delineate the region of the light spectrum that will be eollected into a set number of individual photomultiplier tubes in the multi-PMT array. In this way the META detector can be used as additional detection channels (up to 8 channels). The eonventional single channel detectors (described above) can be used in combination with these META imaging channels. However, all these additional ehannels are limited by bleed-through between individual channels. This will depend on the fluorophore combinations used, but as a general rule up to 3 and possibly 4 different fluorophores can be separated in this manner. Once you attempt to separate more fluorophores the narrow spectral band alloeated to each channel no longer contains fluorescent light unique to that particular fluorophore. The META channel can also be used in a very different mode, called emission fingerprinting, to separate several overlapping fluorescence emission spectra. In this mode aseries of images is collected, which is called a "lambda stack". Each image in the stack corresponds to a single PMT within the 32-PMT array ofthe META detector. A full stack would consist of 32 images (collected as four scans using eight detectors in each pass) covering a defined range of the visible light spectrum. In this way spectral information is collected for each pixel in the image. A narrower region of the light spectrum can be collected by using fewer individual detectors in the multi-PMT array, or the collection step size can be inereased by binning the output of individual PMTs on the array. A computer process called linear unmixing is used to separate out the contribution made to the spectrum from several fluorophores with highly overlapping fluorescence emission spectra. The instrument can utilise stored spectral data to detennine the contributions from each of the individual fluorophores, or you can collect individual spectra from different regions of the sampie or from single labelIed control specimens. The remarkable separation achieved by the META channel me ans that not only can many more fluorophores be imaged simultaneously, but that the fluorophores can all be green! As long as there is a recognisable spectral difference between the Appendix I: Confocal Microscopes Carl Zeiss 389 fluorophores they can be separated by this method. The fmal output from the META channel (after linear unmixing) is not the spectral information, but an individual image from each of the designated linearly unmixed channels. If the META channel is as remarkable as it sounds then why bother at all with conventional channels? The answer is that there are limitations to the use of the META channel. These include issues of detector sensitivity, the limitations of spectral separation, and the constraints of a single pinhole and only one PMT gain and off-set control for the multi-channel META detector. The Zeiss META PMT array detector and associated electronics does produce a detection channel with high sensitivity and excellent signal to noise ratio. However, there is often a discemable difference between the sensitivity of the conventional single PMT detection channels used on the Zeiss LSM 510 instrument compared to the META channel. An apparent lower sensitivity in the META channel may be due in part to differences between the two types of PMT tube, but perhaps more importantly there are other design differences between the two channels that could account for the observed differences. One constraint associated with a PMT array is that the light for each channel is now spread over several PMT tubes instead of being collected by a single PMT tube. When using conventional dichroic mirrors and optical filters, light from a specified region of the light spectrum (which in some cases may be relatively broad) is directed to a single PMT tube. In the case of the META channel the light is collected by up to 32 PMT tubes. When there is an abundance of light the META channel will perform superbly, but as the light becomes much less, the limit of sensitivity of the META channel will be reached before the limit of sensitivity for a conventional channel is reached. Another important difference between the META channel and the conventional channels is that a diffraction grating (with associated loss of light) is used to separate the various wavelengths in the META channel. There will also be some loss of light when using dichroic mirrors and optical filters in the conventional channels, but the amount of light lost will depend greatly on the type of optical filter and the number of filters or dichroic mirrors used. Another important consideration when using the META channel is that there is only a single variable size pinhole and a single detector gain and offset control for the whole PMT array. This means that care needs to be taken to "balance" the fluorescence emission of each fluorophore when using several fluorescent probes. Conventionally this "balance" is achieved by adjusting the individual detector gain levels for each imaging channel. When using the META channel you will need to adjust the fluorescence emission of each fluorophore by adjusting the individual laser line intensities or by adjusting the relative concentrations of the individual fluorophores. If there are significant differences in the fluorescence intensity between individual fluorophores when using the META channel, then using 12-bit instead of8-bit data acquisition will result in a significant improvement in the image from the lower intensity fluorophore. Appendix I: Confocal Microseopes 390 earl Zeiss A very powerful feature of the META channel is the ability to quickly collect the individual reference spectra from a real live sampie, for use in the linear Image Collection Modes on the unmixing algorithm. This would at first appear to eliminate any discrepancies Zeiss LSM 51 0 META between the stored spectral data and your The Zeiss LSM 510 META confocal microscope is sampie. However, many dyes show capable of imaging a wide range of nuorescent spectral changes depending on the molecules using a variety of spectral separation environment of the dye. This techniques. characteristic is exploited in a number of dyes (for example, the changes from Single detector channels - light is directed to green to red fluorescence, depending on individual photomultiplier tubes using conventional the membrane potential of the dichroic mirrors and optical filters. Two individual mitochondria when using a number of channels are available. mitochondrial dyes). Cellular ions, including pH changes also often Single channels + META detector- the META influence not only the level of detector can be used as an additional detection channe/. fluorescence (fluorescein, for example, META detector as a band pass filter - the has a much higher level of fluorescence META channel can be used 10 collect up to 8 different at alkaline pH) but mayaiso alter the regions of the light spectrum. wavelength of emission (acridine orange ranges from green through to red META emission fingerprinting - the META fluorescence, depending on pH and channel can be used 10 collect the emission spectrum of interaction with other molecules). This the sampie and to then use a process ca/led linear variability in the emission spectrum of unmixing to derive the components of the light that can the dye being used could result in be attributed 10 individual nuorophores. misleading results if you use an emission spectrum derived from an inappropriate META + 2-photon excitation fingerprinting - control for use in the process of linear emission spectra are collected using aseries of 2-photon unmixing. However, you can also exploit excitation wavelengths. Linear unmixing is used to derive these differences in florescence emission individual nuorophore components. to obtain valuable information about the local environment ofthe dye. The unique fluorescence separation method used in the META channel is a very powerful tool in confocal microscopy that will allow you to utilise fluorophores that could not be separated by more conventional optical means. The META channel, in combination with the conventional single PMT channels available on the Zeiss 51 0 META, results in a highly versatile instrument for biological imaging. Transmission Imaging The Zeiss LSM 510 META confocal microscope is capable of simultaneous transmission imaging, including Phase and DIC imaging if the correct optical elements are installed on the attached light microseope. The Wollaston prism used for DIC imaging in the Zeiss microscope does not appear to affect the quality of simultaneously collected fluorescence images. The light used to create the transmission image does not pass through the confocal pinhole and therefore the transmission image is not confocal. Appendix I' COnf(lCal Microseopes Carl Zeiss 391 ZEISS CONFOCAL MICROSCOPE CONTROL SOFTWARE The software used to control the Zeiss confocal microscope has a graphical based user interface that displays all of the imaging and microscope parameters in a number of display panels. The software is reasonably easy to use, but the complexity of the instrument does mean that a great deal of information often needs to be presented on-screen - creating some confusion for people not familiar with the instrument. The program is designed to operate on dual screens, with the control panels displayed on monitor land the images displayed on monitor 2. The software can be used in two different configurations, the "expert" mode, where all of the controls are available to the user, and the "routine" mode, where only a seIect group of relevant controls for the particular method selected are displayed. The examples shown on the following pages are all taken from an instrument in which the "expert" mode was selected. The image manipulation and database component of the Zeiss image collection software (Zeiss Image Examiner can be loaded onto a separate computer (available from the Zeiss web site free of charge) to allow easy access to images collected on the Zeiss confocal microscope. Main Control Panel When using the Zeiss software a small concise main control panel is visible as a box on your desktop (Figure A-20). The three buttons on the bottom right ofthe control panel (data collection mode) allow one to readily switch between conventional bright-field or epi-fluorescence microscopy (VIS) and laser scanning confocal microscopy (LSM). If you are using a fully automated light microscope these buttons make all the necessary changes to allow you to either look down the eyepieces of the microscope or to use the laser scanning microscope, without having to manually change optical filters and settings as you move between the two imaging systems. The Zeiss software utilises an excellent filing system for managing your images (accessible via the "file" button, see Figure A-20). This database provides information on microscope and scan head settings when the image was collected, a good thumbnail display ofyour images, and the opportunity to add notes etc to annotate the images. The collection of images on the Zeiss 510 confocal microscope is achieved by activating the "Acquire" button (Figure A-20) on the main control panel. Activating the acquire button will display 8 simple buttons that are used to operate the microscope, the lasers, and the scan head for image acquisition. Detailed information on the menu options available for the "Micro", "Config" and "Scan" buttons are shown in Figure A-21, Figure A-22 and Figure A-23 respectively. Database tor managing images Data collection mode Laser Scanning Microscopy Figure A-20. Zeiss LSM 510 Main Control Panel. The Zeiss LSM main contral panel when using the "expert" mode is a small "box" that sits directly on your computer desktop. This panel gives YOll aceess to all the controls. including mieroscope set up. image acquisition. programming macros. etc. The three buttons on the lower right of the contra I panel allow one to switch between bright-field conventional microscopy and laser scanning microscopy without the need to adjust sliders etc on th e micrascope. The soft wa re used to control the Zeiss canfacal microscape can be configured (during installation) in the "expert" mode. where all parameters are available tor the user to adjust. or in the "routine" mode. where only a seieet group of parameters relevant to the particular labelling being pertllrmed are available. Appendix I: Confocal Microscopes 392 Carl Zeiss Setting up the Microscope and Imaging Channels Single track imaging is the standard method for single or multi-channel imaging (Figure A-21). However, if bleed through is a problem then multi-track imaging (see page 242), where each frame or alternate line is collected using different settings, may provide much better separation between channels. Figur. A-21. Ztiss LSM SIO Im.!:;n!: Chann." S.tup. Thc "Mino" button is. uscd 10 sei up ,he ligh t m,cro,copc für nolh (OI1\CllllOl1al li ght mleros opy .nd ronfoc.1 miero,copy Thc '"("onflg" button opens Ih,' "Contigur~lIon l"Ol1lrol" panel. whlch 15 used 10 .d]uSl Ihe settings for rach 0 Ihe .hanncls ''''''1011) Multl-tra.:k amI mit mein", ,magin~ (an al so Ix' acreS. cd \ 13 th c ronflguratlon cO nlrnl pan,,-I Appendix I. Confoeal Microseopes earl Zeiss 393 Setting the Scan Control Parameters Various scanning parameters, such as the zoom factor, collection box size, scan speed, collection filter etc., all need to be continually adjusted to optimise image collection (Figure A-22). Although these parameters can be obtained from a previously collected image (using the "reuse" button), it is necessary to be always aware of the settings and to continually make adjustments to improve the quality and resolution ofthe image collected . .Ij lSM 510 hp.,' "ode ~ fJoc... Setting the scanning parameters ~ Set parameters for Image collectlon • Frame scannlng adille Scan bOx saft set 10 512 ,,512 pixels line 0< saeen averagong CUlT8I111y set 10 "1" (no averaging) Move scan aroa wilhin fieId 01 vIew (pan). onIy WOf1 Figure A-22. Zeiss LSM 510 - Setting the Scan Parameters. Tbe "Scan" button is used to set the sean parameters for confocal microscopy. On the right hand side of the Scan Control panel are a number ofbuttons used for initiating image aequisition. The "find" button is useful for initially setting Ihe gain for "Iocating" your sampie. When this button is depressed the instrument eolleets a number of images in a relatively fast acquisition mode while varying the deteetor gain, and ifneeessary adjusting the amplifier gain (adjusting the sensitivity ofthe instrument). Onee a suitable gain level has been found. a eonventional single optieal slice image will be displayed - often an exeellent starting point for further adjustments. Appendix I: Confoeal Microseopes 394 Carl Zeiss Adjusting Imaging Channel Settings Each channel in the confocal microscope has a number ofparameters that need to be continually adjusted (Figure A-23). These include the pinhole size, detector gain, amplifier offset (black level) and the laser intensity. These parameters need to be continually adjusted during image collection to maximise the resolution and signal to noise ratio that can be obtained. Each channel (denoted Ch2 and Ch3 in Figure A-23) has an independent set of controls . .'( lSIoi 510 · Experl Mode '.' IV Adjusting the channels • Opbc.lll oke < I) 1 "'" Pride 111 • 2. 1I) ÄlJI Unr" ~ ~r-I- ro ~ ,------t- ~ ~ Exclt.Uon r .58..... rot .!lJ Figur~ ;\-23. üi • LS ~t ~IO - ;\djuninR Imaging bannt i•. r 4n ..... rot .!lJ Thc .. 'can" bUllon opens Iho ··S,·. n COOlror' pand. \\ hore Ihc p .se..... r;-.!lJ umgmg ('on1rols li'U h as P~'1 T goin. pinholc SllC eh: are jLarenne loc'IOO for cach of the rndl\'rdual collectlOn channels (by r 514 ..... ro;- ~ clrckmg on "ChanneL '"). Thc Laser line selectioo and Intensily salti s Appendix I: Confoeal Microseopes Carl Zeiss 395 Image Collection Panel The image collection panel is where the images are displayed during collection, and is usually located on the second screen in a dual screen system (Figure A-24). Individual channels, or a split screen showing all active channels as shown in Figure A-24, are used to assess the quality of the image during collection. The images should be shown as a 1:1 (100% size) ratio when resolution offine structures is important. A merged image (image 3 in Figure A-24) is used to display the individual channels as a multi-coloured overlay. If the "new" button is pressed on the image scan control menu (Figure A-22) a new window similar to that shown below will be created. Use the "reuse" button to select all of the microscope settings from a previously saved image to start imaging with the same conditions as used previously. Figure A-24. Zeiss LSM 510 -Image Collection Panel. A new image collection panel is opened by pressing the "new" button on the "sean eontrol" sereen (see Figure A·21. Figure A·22 and Figure A·23. top right 01' "Sean Control" panel). The images are displayed "live" ",hile seanning in a variety oHormats. inc1uding the "split xy" format shown above. where eaeh individual channe!. as weil as a merged image. are all displayed on the sereen. Individual ehannels ("xy Display") ean also be displayed separately. The image display box size (this does not affeet the image acquisition frame size. see Figure A-22) is adjusted by first selecting the "zoom" button and then seleeting the appropriate image display size. A very useful item found on the image display panel is the "reuse" button. A simple c1ick on this button on any image (even images collected in a previous session. or by another person) will set up al1 01' the image col1ection parameters to those used to collect the original image on display. In this way complex settings from previous experiments can be very easily replicated at a later date. Appendix I: Confocal Microscopes 396 Carl Zeiss Using the META Channel The META channel is a powerful and versatile tool for collecting images on the Zeiss LSM 51 0 META confocal microscope. As discussed above, the META channel can be used in a number of different imaging modes to collect images. These methods are discussed below in relation to how they are implemented in the Zeiss imaging software. META Channel as a multi-Band Pass Optical Filter The Zeiss META detector can be readily used as a multi-channel band pass optical filter by setting the region of the light spectrum that is directed to each ofup to eight separate channels (Figure A-25). In this example only three channels are active (one collecting the green, one the yellow and the third the red region of the light spectrum). Another five channels are available, but have not been activated. In this mode the fluorescent light emanating from the sampie is being separated by a diffraction grating and then the various spectral regions are detected by defined regions of the META multi-PMT array. Using the META as a multi-band pass optical filter does cause potential bleed-through problems, and so this type of multi-channel detection is norrnally used to separate two or more fluorophores that have weil separated emission spectra. Although the sliders used to define the spectral range for individual imaging channels can be positioned such that each channel collects light from overlapping regions of the light spectrum, overlapping settings should be used with caution as they will result in serious bleed-through ~..:;u r J .11---- r wsl 1---• r l ws I Multi-Band Pass Filter r 00511 r (hSSl Figure A-25. Zeiss LSM 510 META Chaunel- Spectral Channel Settings. The META channel can be used to colleet up to eight different regions of the light speetrum as independent imaging channels. In the above example the "green" region of the visible light speetrum (500 to 550 nm) is dirceted to ehanncl I (ChS I). thc yellow region to ChS2 and the red to Chs3. The spectral region directed to each channel needs to be selected earefully to minimise bleed-through between ehannels. These META spectral channels can also be combined with "conventional" imaging channels. The position of each of the imaging ehannels can also be set by using the "Apply to Hardware" button (Figure A-28) on the Lambda display panel (Figurc A-27). Appendix I: Confocal Microseopes earl Zeiss 397 problems for most fluorophore combinations. These META collection channels can also be combined with the two conventional channels available on the Zeiss LSM 510 META confocal microscope (Figure A-21). Separating the light into separate channels by using the META channel as a band-pass filter should not be confused with linear unmixing (discussed shortly), where the fluorescence emission from fluorophores, even with highly-overlapping emission spectra, can be separated into individual imaging channels by computer analysis of the collected spectral image stack. META Channel- Lambda Mode (Spectrallmaging) The META detector can also be used to collect spectral information from defined regions of the light spectrum (Figure A-26). In the example shown in Figure A-26 a region of the light spectrum, from 497 nm ("Start") to 657 nm ("End"), is allocated (by using the on screen sliders) as the spectral region to be collected. The "step" size denotes the width of the spectral region (in this case 10.7 nm) that will be allocated to each individual image. A larger step size of 21.4 nm can be formed by binning two The META Channel adjacent PMT cells in the META multi-PMT array, with even larger step sizes being As a Multi-Band Pass Optical Filter: created by binning more than two adjacent up to 8 individual image channels can be specified. PMT cells. Eight individual spectral steps can be collected in a single pass of the scanning For Spectral Imaging: laser. Further spectral steps can be collected create a wavelength (A) stack, where each image is derived by using multiple passes of the laser (a total of from a narrow (usually 10.7 nm) region ofthe light spectrum. 4 passes of 8 steps each is possible). ~ Extracting Channels: The images collected with the settings up to 8 individual image channels, from defined regions shown in Figure A-26 are displayed using the of the light spectrum, can be extracted from the wavelength or lambda (A) display mode wavelength stack. (Figure A-26, lower panel). In this example ~ Emission Fingerprinting: each individual image displayed is the light unmix individual fluorophores (often with highly collected from a 10.7 nm wide spectral region. overlapping fluorescence emiSSIOn spectra) by The first eight images displayed were separating mixed colour spectra using reference spectra. collected in a single pass of the laser, with the subsequent 8 images (only three of which are shown) being collected during a second pass of the laser. The spectral information collected for each pixel within the image can be used to provide valuable information on the spectral properties of fluorescent dyes within defined subcellular locations. Furthermore, individual image channels from defined regions of the light spectrum can be extracted from the wavelength or lambda (A) stack. Spectral information can also be used in a process called "linear unmixing" to separate fluorophores - even those with highly overlapping emission spectra. Extracting Image Channels 'rom a Spectral Stack The spectral information collected when using the META channel (Figure A-26 and Figure A-27, upper panel) can be separated into defined regions ofthe light spectrum and displayed as individual images (Figure A-27, lower panel). A graphical display of the spectral information within the wavelength or lambda (A) stack is used to help define the region of the light spectrum to be allocated to individual channels. The defmed spectral regions are displayed as individual images (Figure A-27, lower panel), including a merged multi-coloured display if required. This type of spectral separation does require that the fluorophores have relatively non-overlapping spectra, or at least regions of their spectra that are distinctly separate from one another. Overlapping spectra will result in problems of bleed-through as happens when using "conventional" channels defined by optical filters. The smooth graphical display of the spectral data for each ROI (Region of Interest) shown in Figure A-27 is simply a "curve ofbest fit", and should not be interpreted as indicating that the spectral data within the lambda stack produces a "perfect" spectrum. In fact, the "real" data may often be relatively "noisy". At some point the "noise" level is such that a reliable spectrum can no longer be obtained - although a "smooth" curve will still be displayed! Appendix 1: Confocal Microseopes 398 Carl Zeiss Spectral Imaging flgure A-26.:UI LSM 510 META blnnel - L.mbd. Mode. 10 7 nm spectral Tbc META chaMcl can be used. tollee. \ avdeng\h (lambda) w,,'Idow' tot aaCh ,mage image lack. In lhe "Lambda" mode ( e panel I thc righl) a region of lhe lighl pectrum i delineoled by mo ing lhe n feen liders. Tbi region is \hen divided imo a number of •. lep" r pecual "windo\\ .. for colle<:ling the image . p to 2 petlnll "windows" of 10.7 nm can be colle<:,ed (a ,olal runge of 320 nm). Howc\'cr only teps can be collecled per can (henee thc requifemenl ~ r IWO an in Ihi eumple), Tbc pcctrol ' '\\~ ndow '' ize n be doubled, 10 21 .4 nm. or grealer by binning .djacenl PMT in lhe muhi· P~H array, Ea h 6pCCual lep COlle<:led u ing lhc META channel is di played below an individual image (I. display). Each image represenlS a 10,7 nm "window" of lhe lighl pccuum - wilh only 11 out of a lotal of 16 imag col\ e<:ted being di played in Ihi cXllmple . Collectlng a wavelength (I..) stack Wavelength JC display 'r-~.., Display of 11 out of 16 Images collected Display of wavelength (A) stack Appendix I: Confocal Microseopes Carl Zeiss 399 Figure A-27. Zeiss LSM 510 META Channel- Exlracting Channcl•. The lambda (wavelength) stack collected using the META channel (shown above) can be separated out into individual image chaonels as shown below. Image e"lraction is accomplished by first defining regions of interest (ROI) wilhin the image slack .nd displaying graphically (above left) the spectml information for each ROr. Defined speclral regions (circled above left) are used 10 determine Ihe spectml region allocaled 10 .ach image channel (ChS I & ChS2 in Ihis example). The '"Extract channels" button Delined regions 01 the light creates an image display of each channel (shown below). Thc '"Apply to Hardware" spectrum are extracted into button can be used 10 set thc multi-band pass optical filter channels on the META channel1 & 2 channel (Figure A-28). ROt = Region of IntereSI, which can be any defined area of the image slack - including • single pixel as shown above, a defined region (using the ROllools) or even the whole image. Extracting Channels fram Spectral Data Appendix I: Confocal Microscopes 400 Carl Zeiss Apply to Hardware Figure A-28. Zeiss LSM 51 0 META Channel• Apply to Hardware. Thc "apply to hardware" button (found on the lower left 3~ of the display panel in Figure A·27 and Figure A-29 is Saveto used to transfer channel settings to thc META hardware Speclla OB for multi-band pass imaging (Figure A-25) . • NoPa.1. Image channels (ChS1 & ChS2) defined above are applied 10 META channel hardware settings Emission Fingerprinting Spectral image acquisition and linear unmixing are combined in a procedure called "emission fingerprinting" (see Figure A-29) that may be performed both, on- and offline. Emission fingerprinting is capable of separating not only fluorophores that have physically separated emission spectra, but also fluorophores with highly overlapping emission spectra. For example, several green emitting fluorophores can be separated as long as each fluorophore has a sufficiently distinctive spectral characteristic. The process of emission fingerprinting first requires one to collect a fluorescent emission spectrum image stack of the sampie (Figure A-29, upper panel). The spectral characteristics of the fluorophores used to label the sampie are then determined by choosing a region of interest (ROI) and displaying the emission spectrum as a graph (Figure A-29, upper panel where four regions of interest have been marked). The emission spectra of the individual fluorophores can also be determined from a single labelIed control, or taken from a stored spectral database. Linear unmixing is the process by which the spectral data is used to "unmix" the various fluorophores present in the sampie, and to present them as images in separate channels (Figure A-29, lower panel). Emission fingerprinting can also be used to distinguish between different regions of a cell or tissue sampie that show different emission spectra for the one fluorophore. For example, propidium iodide, which has an emission spectrum that shows distinct differences depending on the local environment ofthe probe, can be used with emission fingerprinting to distinguish between DNA and RNA bound dye. Dye bound to DNA has a distinctly different emission spectrum compared to dye bound to RNA, even though the fluorescence emissions of both forms of the dye are highly overlapping. A further powerful feature of emission fingerprinting is that unwanted "background" fluorescence can be spectrally characterised and then removed from the final composite image. Sampies with relatively high background fluorescence, particularly naturally occurring autofluorescence that cannot be easily removed by other means, can be very successfully imaged by using emission fingerprinting. On-Une Emission Fingerprinting Linear unmixing can be used on-line in real time to display several imaging channels. Pre-recorded spectral information from the sampie is used to perform linear unmixing during image acquisition. In this mode images are displayed in separate channels consisting of the emission from individual fluorophores. The spectral information used for linear unmixing is not displayed or saved to disko The ability to do linear unmixing in real time on live sampies is of great value when imaging dynamic processes. In this way cellular processes can be followed on-screen as they are happening in the same way as performing time• lapse imaging using conventional imaging channels. On-line emission fingerprinting also has the advantage of being able to display images in which the background has been removed by linear unmixing in real time. One can use the dynamic removal of background from the image to optimise the image collection of sampies that may be difficult to image using conventional single or multiple channel imaging. Appendix I: Canfocal Microseopes Carl Zeiss 401 Emission Fingerprinting Reg ion 1 y spec1,um .• 2 Region 3 ,. 2 2 1'1) lpec1rum " ~ 1 • 'I) Figure A·29. hiss LSM 510 META Channel- Emission Fingerprinring. Emission fingerprinti.ng is a mcthod by which spectral acquisition and linear unmixing can be used to separate fluorophores based on their speerral eharaererisries. ralher than juS! Iheir degree of speeITal separation. Even f1uorophores wi th highly overlapping emission spectra can be separated in Ihis way. Spectral information can be obtained from thc wavelength (lambda) image stack of the sampIe (as shown above), from a spectral databa e, Or from a lambda image stack of a single labe lied control. Thc process of Linear Unmixing separates each defined speclra, as shown above, into separate image channels (shown below). Appendix I: eonfocal Microscopes 402 Leica Microsystems Leica Microsystems he Leica optics company produces a wide range of high quality optical instruments from cameras to research T grade light microscopes. Leica is particularly renowned for producing superb quality lenses. A subsidiary of Leica, Leica Microsystems Heidelberg GmbH, is responsible for the development and production of the Leica laser scanning confocal microscopes. LEICA CONFOCAL MICROSCOPES The Leica confocal microseopes have been developed over many years into sophisticated instruments that have superb optics, are capable of multi-channel labelling and posses a variety of innovations, such as the ability to spectrally select the fluorophore of interest. All this makes these instruments capable of not only creating excellent images, but also makes them highly versatile for using confocal microscopy as a major tool in the biological sciences. Inverted light microscope Dual screen display I JI ~ Figure A-30. Leica SP2 RS High Speed Confocal Microscope. The Leiea confoeal mieroseopes are arranged with a two monitor display, a programmable panel box (norrnally used to control PMT gain, z-position, zoom ete) and laser control mounted on a workbench next to the vibration isolation table on which thc microseope and scan head is mounted. The lasers are mounted under the main table, with a fibreoptic conneclion 10 Ihe scan hcad. This photograph is kindly provided by Wem er Knebel, Leica Microsystems. Mannheim Gerrnany. Leica Microsystems Heidelberg GmbH Web' huo' (www, Ilt de or hUD'.. i/WWW leica microsvstems comlwebsite/lms nst' Address, Leica Microsystems Heidelberg GmbH Leica Microsystems Inc. Leica Microsystems Pty LId Am Friedensplatz 3 410 Eagleview Blvd. Level 2, Building B, Glade View Business Par 68165 Mannheim Exton, PA 19341 482 Victoria Rd., Gladesvil!e, NSW 2111 Germany USA Australia Phone , +49 (0) 62170280 +1 (610) 3210460 ~61 2 9879 9700 FAX , ~49 (0) 62170281028 ~I (610) 3210426 +61298178358 Appendix I: Confocal Microscopes Leica Microsystems 403 Leica Confocal Microscopes Leica Confocal Microscopes Leica has a long tradition of production of high The lotest confocal microscopeji'om Leica uses 0 specrral imaging quality optics. The company has evolved over del'ice that allo,,"s you to choose the "window" of ..-avelength (colour) for each of up to 4 channels. Furthermore, extremelv many years from the amalgamation of a number of narrow band separation of laser lines Fom jluorescence is possible optical and instrumentation companies, dating back using the AOBSjitrer, os ..-ell os conrinuous attenuation ofthe laser to the middle of the 1800's with Spence Lens and beam using AOTF jiters. The instruments are huilt to customer American Optical Instruments and the "Optical specijications. which may inc/ude the more Iradirional dichroic mirrar / optical jitfer separafion ofjluorescenr light insread of Ihe Institute" in Wetzlar, Germany. In 1990 Wild Leitz spectral separation technique. and Cambridge Instruments merged to become Leica. The headquarters for Leica are 10cated in Leica TCS SP2 AOBS I Full featured visiblelUV confocal ! : microscope (depending on lasers I Wetzlar, Germany, with manufacturing facilities attached) with spectral (SP) I and sales offices in North America, Europe and the , imaging, and replacement of the i Asia I Pacific region. primary dichroie mirror beam I I-______~_s_p_li_tt_er_"_·_ith __ the AOBS lilter. I The Leica confocal microscope is produced by a wholly owned subsidiary of Leica, Leica Leica TCS SP2 RS High-speed confocal!multi-photon , microseopc fur live cell imaging. I Microsystems In Mannheim, Germany, with Utiliscs a velY high-speed re sonant : research laboratories still maintained in the original scanner that allows up to 100 I town, Heidelberg, in which the microscope was frames!sec at 512 x 512 pixels box I developed. size. I 1------"--- - ... - ... ------.' Although the basic optical components of a light Leica TCS SP2 MP : Multi-photon version of the SP2 1 microscope have not changed substantially over I confücal microscope. I the past 10 years, Leica, like the other confocal Leica TCS SPI I First introduetion of the speetral I microscope manufacturers, has introduced a I separation technology in 1998. I number of innovative technologies that have made Leica TCS SL "Personal" confocal microscope: the confocal microscope a very powerful research capable 01' speetral imaging in 2 tool in the biological sciences. Early models of the : channels. Leica confocal microscope were difficult to use Leica ICM 1000 A conloeal microseope designed due to wh at was relatively powerful, but not at all für sm'face measurements in the "user friendly", software. The most recent software materials seienee and industry. from Leica IS based on using a very intuitive Earlier ConfocaI Microscopes graphical interface, where, although the scan head rhe earlier confocal microscope models j;·olJ1 Leica used dichroic controls are all computer operated, the on screen mirrurs and oprical filters to separate the \'ariolls wavelengths of "diai" often resembles the dial or slider and layout jluorescenr light. Thev also used neutral densill'Jilfers tu cuntml the of the manually operated earlier instruments. amounl or luser light reaching fhe sampie. Early in the development of confocal microscopes, Leica TCS NT Introduetion uf NT suttware. Leica introduced a number of innovative Wild Leitz CL SM Compact CLSM seanning head lor developments. This includes the AOTF (Acoustic Aristoplan upright mieroscopes. Optical Tuneable Filter) for attenuating the amount of laser light reaching the sampie and the AOBS Wild Leitz CLSM Inverted conloeal microscope for (Acoustic Optical Beam Splitter) for separating the Fluovert biomedical research. irradiating laser light from the tluorescent light. Another innovative development in the late 1990's Leica TCS 4D Introduction of a smaller sean head in 1992. was the introduction of the Spectral (SP) confocal microscope detection channels. This eliminated the Wild Leitz CLSM First introdueed lor biologieal imaging in 1989. High-resolution need for specific optical filters for each eonfueal mieroscope with tluorophore, with the user being able to simply powerful, but diffieult to use move a slider to define the "window" of light that software running under OS9. could be directed to each channel. Appendix I: Confoeal Microseopes 404 Leica Microsystems Components of the Leica Spectrallmaging Instrument The Leica SP2 laser spot scanning confocal microscope can be configured for visible light, UV light and/or multi-photon microscopy on either upright or inverted microseopes (or interchangeable). DmIp Fluorescent separation using a prism with up to four separate detection channels. The scan head contains all of the necessary optics, pinholes and detectors. Remote lasers are fibreoptic connected. The instrument is a very high-end confocal microscope that has great versatility. Sc. HaId The relatively large scan head is build onto either an inverted or upright Leica light microscope. AOBS: In the AOBS model the selection of the laser line used for exeitation is performed by the "Aeoustic-Optical Beam Splitter" filter - wh ich allows for very narrow separation of laser lines from emitted fluorescence. Dichroic mirrors: In instruments without the AOBS beam splitter, various dichroie mirrors are available. Spectral prism: Returning fluorescent light in the "SP" model is spectrally separated using a prism. Carefully positioned computer operated "slits" are then used to direct a user-defined region of the spectrum to each of up to four photomultiplier tubes. Variable collection slits: Light cntering each of the photomultiplier tubes must first pass through a variable slit, which controls the region ofthe spectrum that is detected by that particular channel. rfl Variable size confocal iris: Single computer controlled variable size "pinhole". Barrier filters: Computer-controlled wheel of removable barrier filters is available for selection of suitable wavelengths in models that are not equipped with spectral selection ofwavelengths. Light detectors: Photomultiplier tubes (the number depends on the number of detection channels available - up to 4, depending on the model), are housed within the scan head. Scan mirrors: X & Y fast galvo sean mirrors with zoom, bi-directional & sean rotation capabilities, U assembled into a single unit ealled the K-scanner. The controller box contains the eleetronics associated with controlling the scan head and the collection and digitisation ofthe signal from the photomultiplier tubes. Lasers: Wide range of lasers can be attached via fibreoptic connection, including the Krl Ar, Ar-ion, HeNe, 405 nm violet and red laser diode, HeCd and single line Kr-ion. UV and Ti:sapphire multi• photon lasers can be attached to suitably configured instruments. Laser power: Adjusting the laser power knob sets the maximum power available - the aetual power level at the sampie is determined by the AOTF setting. AOTF laser attenuation: Computer controlled AOTF filters eontrollaser intensity at the sampie. The relatively large scan head and assoeiated opties ean be attached to either an upright or inverted Leiea microseope, but this is usually carried out by teehuical experts from Leica. The scan head cannot be readily attached to microseopes from other manufaeturers. Leica is a manufacturer of light microscopes, and lenses as weil as the confocal mieroscope sean head. Multi-channel fluorescence, backscatter (reflectanee) and transmission. Box sizes up to 4096 x 4096 pixels. Line, ROI, zoom, panning and rotated scanning. Time lapse imaging. Simultaneous and • sequential frame or line multi-channel scanning. 12-bit data aequisition and processing saved as 8-bit or 12-bit image files. Single ehannel photomultiplier deteetor. Capable of bright-field, phase and DIC imaging, depending on the optieal settings available on the light microseope. Programmable manual eontrol knobs (panel box) used to control all essential acquisition parameters. c:ompu.r High-end PC computer to control the confoeal microseope, the settings on the light microscope if a --~- - ' fully automated mieroscope is installed, and to eollect the images. Dual computer sereens are used to display both the images as they are collected and to display the eontrols for scanning and chan ging microscope settings. Soft'I The Leica software is used to control the scan head, eollect the image, and some image manipulation and 3D reconstruction. A limited version of the Leica software (LCS Lite) provides basic image manipulation, and can be loaded onto another computer without incurring any further costs. ~Iodds Multi-channel spectral separation (SP) -- detailed specifications (number of channels, speetral verses eonventional optics, AOTF, AOBS etc) are required for assembly to customer specifieations. Multi• photon system available. High-speed video rate resonant scanning system available. Appendix 1: Confocal Microscopes Leica Microsystems 405 Leiea rcs SP2 Confoeal Mieroseope The Leica SP2 spectral separation confocal microscope is a highly versatile instrument based on the use of a prism to separate the fluorescent light into various regions of the light spectrum. This instrument can be used as a multi-channel confocal microscope by directing different spectral regions to up to four independent detection channels. The instrument can also be used to collect spectral information for each pixel within the image, which can then be used to separate highly overlapping fluorophores by a process called linear unmixing. variable slit & PMT fOf each of 4 channels Expanded view of variable slit for selecting region of spectrum Fibre optic conneded lasers Extemal multi-photon detedOfS Transmission detector Figure A-31. Leica SP Confocal Microscope Scan Head. Leica spectral selection (SP) confocal microscope scan head uses a prism to separate the retuming fluoreseent light into separate colours. Computer controlled variable slits are used to seleet the region of the light spectrum directed to each of up to four detection channels. In this example the highly efficient AOBS device is used to separate the laser excitation light from the retuming fluorescent light. This figure is adapted from a diagram kindly supplied by Wemer Knebel. Leica Microsystems. Mannheim Germany. A diagrammatic representation ofhow the Leica SP spectral separation scan head works is shown in Figure 3-11 on page 82. Leiea rcs SP2 Sean Head The latest confocal microscope scan head from Leica Microsystems contains a number of highly innovative technologies (Figure A-31). These includes the Acoustic Optical Beam Selection (AOBS) device to very efficiently separate the irradiating laser light from the retuming fluorescent light, a prism to spectrally separate the fluorescent light, and computer controlled slits to direct specific regions of the light spectrum into up to four independent channels. Appendix I: Confocal Microscopes 406 Leica Microsystems The relatively large scan head contains all of the necessary optical components for confocal microscopy. Only the lasers, which are connected by fibreoptics, and the extemal detectors used in multi-photon microscopy, are located remote from the scan head. Various lasers can be readily connected via three main laser entry ports. One port is for connecting visible light lasers (a wide range of lasers can be connected to this port). In the example shown in Figure A-31 the AOBS beam splitter (described in more detail below) is used to split the incoming visible light from retuming fluorescent light from the sampIe. However, the IR laser used for multi-photon microscopy, and UV lasers are connected via separate connections that utilise dichroic mirrors to separate the irradiating light from the fluorescent light. Each laser port contains an excitation pinhole, but there is only a single very small (micron sized) v-shaped variable sized confocal pinhole in the Leica sr scan head. This pinhole is located before the fluorescent light is separated into different spectral regions, which greatly simplifies pinhole alignment, but does mean that the pinhole size cannot be matched to the wavelength oflight being examined in multi-Iabelling applications. The Leica confocal microscope scan head assembly is based on customer specifications at the time of purchase, which means that Leica confocal microscopes will be found to contain a range of features and innovations that vary between individual instruments. Acoustic Optical Beam Splitter (AOBS) The Acoustic Optical Beam Splitter device (described in more detail on page 78 in Chapter 3 "Confocal Microscopy Hardware") on the latest Leica confocal microscopes allows for highly efficient separation of the irradiating laser light from fluoreseent light returning from the sampie. The AOBS element is an optional replacement for the primary dichroic mirror used in the Leica sr confocal microscope. The advantage of the AOBS element is that a very narrow band of light (essentially a single wavelength, for example 488 nm) can be selectively directed to the sampie - with the full spectrum of returning fluorescent light (except for the very narrow band of 488 nm light) being allowed to pass through the device to the detectors in the sc an head. Up to 8 individual laser lines can be high-speed switched and separated from the returning fluorescent light. The AOBS element results in significantly less of the fluorescent light being rejected, resulting in an increase in sensitivity ofthe instrument. Furthermore the AOBS device, being capable ofvery high-speed line switching, means that images can be collected using different excitation Iines for each alternate scan line in the image. Laser line switching can also be achieved by electronic switching ofthe AOTF filters used to attenuate the intensity ofthe laser light, but the AOBS element can both select out specific narrow bands of light and switch between multiple bands at very high speeds in the one optical device. The AOBS filter has the additional advantage of being readily programmable to select out the wavelengths of choice if a new laser is installed - without the need to install additional optical filters or dichroic mirrors. Spectral Separation The innovative method of spectral separation of various regions of the light spectrum in the Leica confocal microscope results in a highly versatile instrument that has a simple and intuitive means of separating different wavelengths of light in multi-labelling applications. This includes on screen software that mimics the physical settings ofthe variable width slits (see Figure A-34). Spectral separation of the light spectrum using a prism is a very weil known phenomenon, but where the Leica instrument excels is the way in which up to four detection channels are designed to collect specific regions of the light spectrum without any "dead" area between the channels. The way this is achicved is by using computer controlled variable width slits that have mirrored surfaces. The position and width of a slit determines the region of the light spectrum that is directed to that particular channeI. Light that is excluded from the slit is reflected from the mirror surface of the slit to a second mirrored slit where a further region of the light spectrum can be separated - up to four such channeIs may be available on the Leica SP confocal microscope, depending on how many channels were specified by the customer when the instrument was assembled (see also page 81 for further discussion on how this spectral separation technique works). Spectral separation can be used for routine multi-Iabelling applications, although a simple dichroic mirror system may be just as efficient at separating the fluorescence emission of weil separated fluorophores. The Leica software does provide a number of pre-programmed and programmable menu se\ections that will set the slit position and width for a wide range of common fluorophores. However, one of the more powerful features of the flexibility of the Leica system of spectral separation is the ability to readily change the position and width of each individual Appendix I: Confocal Microseopes Leica Microsystems 407 collection channel while imaging. This allows one to adjust each channel for optimised collection of multiple labels even when using unusual fluorophores or when attempting to separate specific spectral regions for a single fluorophore. The spectral separation technology of the Leica confocal microscope scan head can also be used to determine the spectral characteristics of the image (lambda scanning). In this technique an image is collected by using a slit width covering a narrowly defined portion of the light spectrum, with subsequent images being colleted by using narrow windows of shorter or longer wavelengths of light. In this way aseries of images that specify the spectral characteristics of each pixel within the image can be collected. Spectral information of individual fluorophores carries valuable information on the local environment of the dye, but perhaps more importantly spectral imaging can be used to separate fluorophores with distinct but highly overlapping spectra. This process, called linear unrnixing, which was pioneered by Carl Zeiss using the META detector (see page 388), is now available on the Leica SP confocal microscope. UV Excitation An Ultra Violet (UV) emitting laser can be readily connected to the Leica confocal microscope scan head. However, the UV laser is a difficult laser to use in confocal microscopy, both due to the destructive power of the relatively high-power UV light and the optical difficulties of dealing with wavelengths that are not ideally suited to the design of the scan head or light microscope being used. With the aid of special UV correction optical elements the UV (and also 405 nm violet laser line) focus is shifted into the position for visible light. This ensures that the emission light passes through the confocal detection pinhole. Multi-Photon Excitation The Leica confocal microscope is capable of multi-photon excitation by the attachment of an ultra-short pulsed infrared laser. Multi-photon excitation is particularly suited to deep tissue imaging as the infrared light can penetrate much deeper into living tissue than can visible light lasers. The multi-photon laser is also capable of exciting many commonly used UV dyes - making the multi-photon microscope an alternative to the UV confocal microscope. Multi-photon imaging does not require a confocal pinhole (see page 94 in Chapter 3 "Confocal Microscopy Hardware") and so a more efficient means of collecting the fluorescent light is to attach extemal detectors (see Figure A-31). These detectors are housed outside the scan head, and collect fluorescent light that has not passed through the confocal pinhole or back through the scanning mirrors. Extemal detectors result in a significant increase in sensitivity ofthe instrument when multi-photon imaging. Transmission (Die) Imaging The Leica confocal microscope is capable of collecting excellent transmission images, which can be combined with single or multi-channel fluorescence images. The transmitted light detector is located after the condenser in the light microscope, in which case care should be taken to correctiy adjust the condenser as this is now part of the optical system ofthe microscope (see 42 in Chapter 2 "Understanding Microscopy"). Excellent DIC images can be collected by using properly installed and adjusted DIC optics on the light microscope. However, if high-resolution DIC and fluorescence images are required, collect the two images sequentially by removing the Wollaston prism from the optics path when collecting the fluorescence image. Otherwise you will find that the fluorescence image will consist of a slightly offset "double" image - this is only obvious when you image at high zoom with a high NA lens, but will detract from the quality of the fluorescence image even at lower zoom levels. The position of the slight off-set is not affected by making adjustments to the Wollaston prism or by removing the polariser or analyser - removing the Wollaston prism is the only effective way of eliminating this artefact. DIC can be implemented in a number of re la ted, but technically different ways, not all of which have an effect on the quality of the fluorescence image. Appendix I: Confocal Microseopes 408 Leica Microsystems LEICA CONFOCAL MICROSCOPE CONTROL SOFTWARE The software used to control the Leica confocal microscope has a particularly easy to use interface - although, as with all confocal microscopes, the complexity of the instrument does mean that the software can at first appear daunting to the user. The highly graphical interface, and the programmable desktop knobs (programmable panel box), mean that most controls are within easy reach. All of the "buttons" available on the control screen can be "hidden" or brought out by using the settings in the "Tools" menu - so if a "button" has gone missing, try looking in the customising tools menu. The following pages provide abrief introduction to the software used to control the Leica confocal microscopes. For a more detailed explanation please consult the Leica confocal microscope manual, the on-hne help, or perhaps best of all ask somebody who is already famihar with the software. The Leica confocal microscope software runs under Windows NT/XP. If you have been set-up as a user on the system, the password log-in is designed to allocate you a specific folder for images and to allow you to recall previously saved microscope settings (imaging methods), but is not intended as a security system for your files. Main Control Panel- Monitor 1 The main control panel (Figure A-32) is used to control the settings on the confocal microscope, and to save images under a specified folder name. The lower part of the control panel contains aseries of "buttons" that are used to control or access submenus that are used to operate the instrument. The "Continuous" scan button is used to provide continuous scanning while locating the sampie and adjusting the instrument. This button does not result in any ofthe images being saved to a file. To save images you will need to press the "Single Scan" or "Series" buttons, which will scan and save the image as a TIF file (saving it to a temporary folder, until you have specified a speeific folder name). "Single Scan" will collect an image using the settings specified on the control panel. These settings inc1ude the pinhole size, speed, direction of scan etc, but also whether frame or hne averaging has been specified. For example, if frame averaging is set to 8 then "Single Scan" will collect a single image, consisting of 8 scans that have been averaged. The "Series" button also collects an image using the pre-set parameters and saves the images to a file. The "Series" button is used to collect a z-series or a time-series image set. As the images are saved as single "TIF" image files (one file for eaeh image in the series, or a single image file when not collecting aseries), the Windows thumbnail display will show each of the images in the folder. These individual "TIF" files can also be readily accessed through most image processing programs, ineluding Photoshop, if you save the images as 8-bit files rather than 16-bit files. Information on instrument settings is all conveniently displayed in a "window" as shown in Figure A-32. This information is saved with the image file, but can only be readily accessed when using the Leica software to look at the saved images. However, the information is saved as a text file in the folder containing the images and so can be accessed with a simple text editing program ifnecessary. Y ou can specify your own file folder name for the images by saving the file - or you will be prompted for a name when you attempt to elose the file or shutdown the computer. Leica Lite A free, cut-down version of the Lelca software that can be used to open Leica image files Appendix I: Confocal Microscopes Leica Microsystems 409 Save your images under a folder name Information on instrument settings _PH'l Continuous imaging - but Colleet a sngle i image Colleet aseries images are NOT saved (automatically saved) (time or z-seetion ) Figure A-32. Leica SP2 Screeu Oue - Main Control Panel Leica uses two computer screens; sereen one (above) being used to display the control panel for operating the confoeal mieroseope. This partieular contra I panel is from a Leiea eonfoeal microscape that has speetral imaging and the AOBS beam splitter for separating the laser !ine from the emitted fluoreseenee. In this eontrol panel a number of images (same with multiple ehannels) have all been saved under the folder name "ExperimentS". The images are saved as individual "TlF" format files that ean be readily accessed by most image processing software. Appendix I: Confüeal Microscapes 410 Leica Microsystems Changing the Desktop Knob Functions. The physical desktop knobs (programmable panel box) provided with the Leica confocal microscopes can be programmed to control a variety of useful functions. The default settings are for the first knob (from the left) to control the gain on PMTI, and second the ga in on PMT2 etc as outlined in the control panel below (Figure A-33). You can readily change these default settings by clicking on any one of the knob labels displayed on the bottom of the main control panel - and then choosing the control function you want the knob to perform from the list provided. "Smart Gain" is a useful function to apply to the first control knob - then, whichever channel is highlighted with the mouse (on the imaging screen) will be controlled by this knob. This allows one to control the gain, for example, of all four imaging channels by using the one physical knob - freeing up the other knobs for other functions. ------=-,,, ClIO 0tbIl '"I I provided wilh Ihe Leic. confocal mi croscopes can bc programmed 10 perform whichcver funclion you _"'Il prefer (c hoose from Ihe lisl .vailable by c1icking on "" ..., 1 Ih e bUllon icon on Ihe bOllom of Ihe main conlrol ClIfUtcPH14 menu bar as shown in Ihis diagram). OfiMt pnl NDOl Off '"1 MK)) (Wf.. PM r r,... __.1 _1-'1 New knob - fUnction~ " - ----<$-. - Im con ,roI "rh'ghhgh",d ,·haM< I I _Oll... t_ c.Iipn- • - \ Appendix I: Confoca! Microscopcs Leica Microsyslems 411 "Beam Path Setting" Control Panel One of the best features of the Leica confocal microscope controlling software is the "Beam Path Setting" control panel shown below (Figure A-34). The great feature of this panel is the graphie display of the colour spectrum, including an overlay of the emission spectrum of the fluorophore you are using. Simply "grabbing" the ends of the slider windows and expanding or narrowing the collection window is a very simple way of modif'ying the regions of the light spectrum that are directed to the individual channels. Aseries of pre-set parameters can be obtained by c1icking on the fluorophore setting displayed in the top right panel (in this example the "U" or "user" settings are displayed - these are the settings that you can modify and save under your own file name. There is also a set of "L", or "Leica" settings that are installed with your instrument and can be called up as required but cannot be modified by the user). The LUT (colour display) used for each channel can be easily changed (including the transmission channel) by c1icking on the graded colour display below the PMT channellabel and choosing the colour gradient of your choice. Don't forget to "tick" the "active" box when you want data collected from a particular channel (only PMTl and 3 channels are active in this example). Figure A-34. Leica SP2 Beam Palh Settings. Monitor I - beam path settings screen is used to set the "window" of light directed 10 each channel. To display this panel dick on the "beam" icon in the lower bottom len of the main control panel (see Figure A-32). Please note: The fluorophore spectra overlaid on the coloUf spectrum in this panel is taken from stored spectral data - and are not spectra derived from your sampie. Use these spectra only as an approximate guide to the spectral emission of the fluorophores used to label your sampie. Appendix I: Confocal Microscopes 412 Leica Microsystems Control "Buttons" 'or Image Collection The main control "buttons" for image collection (Figure A-35 and Figure A-36) are found in a group, usually at the bottom of the control panel screen or within the "Beam Path Settings" panel (on monitor I). The buttons displayed and the layout used can be readily altered by the user (from the "Tools" options on the top of the control panel) - so you may find the instrument you are using has a somewhat different layout. The most common buttons are explained below in Figure A-35, and on the next page in Figure A-36. Figure A-35. Leica SP2 Main Control Panel. Tbe main control panel is nonnally located on tbe lower part of tbe screen, and contains a variety of "buttons". Tbe common "Acquire" buttons used are shown above. However, you may find tbat YOUf instrument bas been programmed to displaya slightly different set ofbuUons. See Figure A-36 for a description offurther buttons that can be displayed. Zoom control on the Leica confocal microscope can be achieved in a number of ways (Figure A-36). A convenient way to make small changes in the zoom is to use a programmed physical knob on the desk top (Figure A-33). However, better control over the amount of zoom is obtained by selecting a specific zoom level using the "Zoom" button on the control panel (Figure A-36). An excellent way to zoom in on a specified area of the image, such as a single cell, is to use the "Z.In" button to draw a box around the region of the image of which you wish enlarge to fill the imaging box. Take care to make sure you have set the "Obj" button to reflect the objective in use, otherwise you will find the one Airy disk pinhole setting and scale bar display are not correct. However, the objective setting will be taken care ofwhen you change objectives ifyou are using a fully automated light microscope. Another button that has an important impact on the quality of the images collected, but is often overlooked, is the "Expan." button, which controls a beam expander lens. Changing the setting for the be am expander will have a large impact on both the resolution and sensitivity of the instrument. The beam expander must be correctly set for each objective - simply try changing the beam expander to find wh ich setting is best for your particular lens and application. The "Sc an" button is used to switch between bi-directional and single direction scanning. However, when switching to bi-directional scanning take care to adjust the "Phase" by using a programmed desktop knob to bring the alternate sc an lines back into alignment. Appendix I. Confocal Microscopes Leica Microsystems 413 Im_bo•• 1D (on po.els) .....toll· )) '.-IM ' mavnll~ Be.m path "tI.nll" "" .... 1 ~.., COnv8nlionai horizonlal imege. 1 'I" .• ". ~ ... 1014·1111 : o Plnh Change "expansion "ni" 10 svil the Jens In usa - changing Ihis Jens wil Single or bI-dlrKtlonal Icannlnll: Use have a arge l impact on the resolution -- "Phase- adjustmenl (...se physical knob on and sensUMty 01 the instrument 114.116 ~'" computer desk 0< abo.... button) to aKgn bklirectlonal scans ' Scan speed -- ... lOOHl Approx. 1sedscan 10. a ... 512 • 512 pillellmage Sei pinhole 10 1 Airy d' k _ slze 10< optimal resoIuIlon with good sensltivity 1000 Hl Figure A-36. Leica SP2 Control Buttons This figure is a continualion of Ihe descriplion of some of Ihe common "acquire" buttons located on the main contral panel (see also Figure A-35). These buttons can be displayed in the main control menu bar, or altematively, they can be displayed in the "Beam Path Settings" panel (as shown in Figure A-34). Which buttons are displayed and their layout on the screen can be changed by the user .• whieh means you will find the instrument you are using will have a somewhat different display to the above. Warning: - be patient when using the Leica software. Make sure each operation is completed before you click on the next button - or you may ·crash" the program! Appendix I: Confocal Micrascopes 414 Leica Microsystems Image Collection Pane/- Monitor 2 The image collection display panel is normally located on monitor 2 (Figure A-37), but this display panel can be placed on monitor 1 if required. Each experiment is displayed as aseparate window on this screen. To dose and save a particular experiment simply dick on the normal windows dose function (the top right hand cross) and you will be prompted for a file name (if you have not already specified one) and whether you wish to save the image - be fore the window is dosed. Take care to specify 1: 1 image display (Figure A-37) using the "Display" button when high-resolution imaging. In this way all of the image data collected will be displayed on the screen. Displaying the images as "Auto" is convenient for allowing several images to be displayed on the one screen in multi-labelling applications, but don't forget that in this display mode not all ofthe image pixels collected will necessarily be displayed. Figure A-37. Leica SP2 Screen Two - Image Display. The second computer screen is used to display the images as they are collected. The images can be displayed in a variety of sizes. depending on whether seeing all ofthe pixels (the I: I view) or thc entire image is more important. You can set the LUT colour display used tor thc images du ring collection by changing the settings on thc individual channeIs on the "beam path settings" panel (Figure A·34). Appendix I: Confocal Microseopes Nikon 415 Nikon Instruments he Nikon optics company produces a wide range of high quality optical instruments, including a variety of T research grade light microscopes and superb lenses. The Nikon research grade light microscopes are used for both the Nikon confocal microscopes, and as the main microscope used by Bio-Rad for attachment to the Radiance or MRC scan heads. NIKON CONFOCAL MICROSCOPES The Nikon confocal microscopes use the established technology of dichroie mirrors and optical filters to separate the fluorescent light into individual imaging channels. The Nikon instruments are aimed at the "personal" user, where high quality optics for specific applications is perhaps more important than the greater versatility ofthe more complex (and more expensive) models available from other manufacturers. Inverted light mlcroscope Optlcs/detector unlt I Figure A-38. Nikon CI Confocal Microscope. The Nikon Cl eonfoeal mieroscope eonsists ofa small sean head (shown here direetly attached to the side port ofa Nikon inverted research grade light mieroseopel. and a fibreoptie conneeted optics/deteetor unit and a fibreoptie eonneeted laser launch unit. The Nikon Cl eonfoeal microscope is designed on a modular basis for ease of instrument upgrading and transfer bet"een microscopes. Nikon Instruments \Veb' www a,e nikon co in inst Biomedicalclindex htm Addrt'~s: Nikon lnslech Co .. LId. Nikon Instruments [nc. Parale Mitsui Bldg .. 8. 1300 Walt Whitman Road Higashida-eho. Kawasaki-ku. Melville. Nm York 11747-3064 Kawasaki. Kanagawa 210-0005 USA Japan Phont>: +81-44-223-2167 + 1 (63 1) 547 8500 -81-44-223-2182 Tl (631) 547 0299 Appendix I: Confoeal Microscopes 416 Nikon Nikon Confocal Microscopes Nikon "Personal" Confocal Microscopes Nikon is a major manufacturer of a wide range of optical based instruments, ranging from conventional cameras to The Nikon con/ocal microscopes have been designed with the "single" laboratory user in mind rather than the large digital cameras and a variety of high quality light multi-user facilities where more versatile, but signiticantlv microscopes. higher cost, con{ocal microscopes are often installed. Nikon also manufactures optical lenses for use in a Cl Digital Eclipse Three fluorescence channel variety of instruments, from cameras to research "modular" confocal microscope, plus transmission channel, that microscopes. Nikon produces a wide range of very high can be readily upgraded after quality microscope objectives, inc\uding non-immersion purchase and installation. The or "dry" objectives as weIl as oil, water, glycerol and confocal microscope is designed multi-immersion objectives. Nikon produces a superb on a modular basis, which includes interchangeable optical "air" objective with relatively high NA and a very long filter blocks for use with various working distance - with an adjustable coIlar to aIlow for dyes. The small and lightweight various differences in coverslip thickness or to corrcct fibreoptic connected sc an head for spherical aberration within the sampie. can bc readily mounted on a variety of microscopes. Nikon manufactures exceIlent "research grade" upright and inverted microscopes that are used in many laboratories around the world. The Nikon confocal PCM 2000 Manually operated "personal" or microscope sc an heads can be attached to a variety of laboratory based confocal microscope with two microscopes, inc\uding microscopes from other fluorescence channels and a manufacturers. The smaIl scan head of the Nikon single transmission channel, or confocal microscope makes moving the scan head from, three fluorescence channels for example, an upright to an inverted microscope, a without transmission imaging. The smalI, easily moved, sc an relatively simple matter. head can be attached to either an Nikon research grade light microscopes are often the upright or inverted microscope. Manually controlled pinhole size microscopes of choice for attachment of the Bio-Rad selection and dichroic mirror and confocal microscope scan head when purchasing a Bio• optical filter changes. Rad confocal or multi-photon microscope, The Nikon laser scanning confocal microscopes are Nikon High-Speed Confocal Microscope designed as a number of separate "modules" that can be This laser scanning con/ocal microscope can achie\'e image readily upgraded or replaced. The scan head is relatively acquisition rates as high as 30 frames per second. This smaIl, with most of the optics and the detectors being instrument is designed as a high-speed confoeal microscope for studies involving fast cellular processes such as located in a fibreoptic connected "optics box" that is intracellular calcium ion changes. placed on the bench near the microscope. RCM 8000 High-speed dual fluorescence The Nikon confocal microscopes are intended as a lower channel confocal microscope cost alternative to the more expensive confocal capable of imaging 30 frames per microscopes available - without compromise to the second with an image size of 512 x 483 pixels. Specially dcsigned optics of the instrument. The cost savings are due to the for ratiometric imaging using more limited versatility of the instrument - which is visible or UV laser lines. Real often fine for a "personal" confocal microscope where a time ratiometric display of image laboratory often has a relatively c\osely defined use for data is possible at high-speed acquisition rates. the instrument. Appendix I: Confocal Microscopes Nikon 417 Components of the Nikon C1 Confocal Microscope The Nikon CI is a visible light laser spot scanning confocal microscope that can be attached to either an upright or inverted light microseope. DetIpl The CI confocal microscope uses dichroie mirrors and optical filters in the form of "filter blocks" that are used in conventional epi-tluorescence microscopy. The instrument is intended as a "personal" confocal microscope that ean be purchased by individuallaboratories. Hmd The Nikon CI sean head is a small and compaet design that is eonneeted to the controller box and lasers via fibreoptie eoupling. The eompaet design means that the Nikon CI sean head ean be readily transferred from an upright to an inverted microseope. The scan head optics are faetory aligned, although minor adjustment ofthe alignment ofthe sean head into the mieroseope will be required. Dichroic mirror: Mountcd inside the CI sean head is a dichroie mirror filter slider, that ean be • manually ehanged, for initial separation of irradiating laser light from retuming tluoreseent light. Changing the confocal iris size: A eomputer-eontrolled wheel eontaining three different pinhole sizes and a 4" "open" pinhole setting is mounted inside the scan head. Fibreoptic pickup: The tluoreseent light, after passing or being detleeted by the primary dichroic mirror mounted within the sean head, is transmitted to the opties box by fibreoptic cable. Scan mirrors: X & Y rast galvo sean mirrors with zoom capabilities. The retuming tluoreseent light is transferred from the sean head to the controller box via a fibreoptie eable. The light then passes through suitable dichroic mirrors and barrier filters, and into the PMT tube. The digitised image is then transferred to the computer. Barrier filters: Filter cubes for a variety of tluorophore combinations can rcadily be changed (manually) to match the particular tluorophore combination used. These filter eubes are physically the same as those used in the Nikon epi-fluoreseence microscope, but often with special dichroic and • optical filter combinations for labelling applications particularly applicable to confocal microscopy. Light detectors: The small compact optics box can be readily swapped over from the standard two photomultiplier tube detector unit to a three detector unit when such an upgrade is rcquired. I..-, Lasers: A wide range of lasers ean be attaehed via fibreoptic connection, including the Kr/Ar, Ar-ion, • '-----"'= HeNe, 405 nm violet and red laser diode and HeCd (up to three lasers at a time). Neutral density or AOTF filters: Aseries of neutral density filters (or optional AOTF filters) are loeated within the controller box. The appropriate filter is manually selected (ND filters) or computer adjusted (AOTF filters) to adjust the amount oflaser light that reaehes the sampie. Laser line selection: Suitable filters ean be placed in front of the laser beam before the laser light enters the fibreoptic cable for transfer to the sean head. A computer controlled shutter is used to seleet the appropriate laser. The sean head ean be attached to either an upright or inverted microseope. The scan head is designed to be fitted to Nikon research grade mieroscopes, but the scan head can be readily connected to other microseopes - perhaps an existing research grade microscope you already have ...... Mods Multi-channel tluorescenee, backscatter (retlectance), transmission. Box size user selectable up to 2048 x 2048 pixels. Zoom and panning. Time lapse imaging. 12-bit data acquisition and processing. A separately supplied transmitted light detector with bright-field, Phase and DlC imaging capability if the microseope is fitted with suitable optics. ~o.roI The Nikon CI scan head is partially manually operated (scan head dichroic mirror sliders are changed by hand), with computer control of image acquisition parameters. ~ High-end PC computer to collect images, adjust PMT voltages and to move the z-position when L-__~_ collecting z-sections. However, many functions within the scan head, and the optics box are operated manually. A single large computer screen is used to both display the images as they are collected, and to display the controls for scanning and chan ging microscope settings. Nikon acquisition and analysis software (including VBA macro language) under Windows 2000/XP. A modular design allows this instrument to be upgraded to incorporate more channels (up to 3) or to separate specified regions ofthe light spectrum by purchasing different optical filter blocks. Appendix I: Confoeal Microseopes 418 Nikon Nikon C1 Scan Head The Nikon Cl confocal microscope manufactured by Nikon is based on the well-established technology of dichroic mirrors and optical filters, with a manually controlled sc an head that offers excellent optics for a very affordable price. The Nikon Cl sc an head (Figure A-39, upper photo) is small and robust, allowing for easy transfer from upright or inverted microscope. The sc an head houses the scanning mirrors, confocal pinhole wheel, primary dichroic mirror, and associated optics. All other optical components, inc1uding the dichroic mirrors and optical filters used to separate various regions of the light spectrum are housed in the fibreoptic connected Optics/Detector Un it. The confocal pinhole size is altered by changing the position of the pinhole wheel, wh ich contains a range of pinhole sizes, inc1uding an "open" no-pinhole position. Optics/Detector Unit Various regions of the light spectrum are se para ted and directed to individual detection channels within the optics/detection unit (Figure A-39, photo graph on the left). This un it can have up to three separate detection channels (3 photomultiplier tubes and 3 filter blocks). The optical filter blocks are physically the same as those used in the Nikon epi-fluorescence wide-tield microscopc - although Figure A-39. ikon CI Confocal Microscope. The small robust Nikon C I eonfoeal mie roseope sc an head is shown abovc allachcd to the side port of a Nikon inverted microseope. The CI opties/deteetion unit (shown on the Jen) houses the neeessary opties for splitting the light speetrum into v"rious regions using Nikon Ouoreseencc mie roseope optieal lilter eubes. The laser launeh uni t (shown below) can house up to three independent lasers. with the light being transfcrred to thc sean head via libreoptie eonneetion . you may require specialised optical filter and dichroic mirrors. depending on the fluorophores being used. The filter bocks can be readily changed for optimal detection of various fluorescent dyes. Laser Launch Unit The laser launch unit (Figure A-39. lower photo) is an integrated la er platforrn on which several different lasers can be mounted. The laser launch un i! shown in Figure A-39 has an air-cooled argon-ion single-Iine la er and a green He e laser installed. Laser line selection and intensity control are provided by the unit. Laser Iines from the different lasers can be combined if required and then transferred 10 the scan head by fibreoptic connection. Appendix I: Confocal Microscopes Nikon 419 NIKON C1 USER INTERFACE I Start scanning k.... ~:= Sngj< Z ao:Jo. • "ne Se.> Seit Objective in use ---+- Z FA Setting up the z-steps Laser select i o~ I 10s :::J (Ci [C2 fC3 [Ci .- Select active detection channels 1)10\1 o ~ d~J Ga;n se";n9$ { : g.>I'2 o .!ll 2.J.!!:!l 1)10\3 ro ~ iJ~ -,ro~ ~ ,~ c Image colour display setting tor each channel Display brightness and contrast adjustment r O.... L..... ra ------~j; ~------s...... ra ------~J~------G 110 ------~j~------ Figure A-40. Nikou Cl Main Control Panel. The Nikon CI confocal microscope is controlled from the above panel. All scan head and laser controls are available from this one control panel (which is usually displayed alongside the imaging panes). The Nikon Cl computer interface is usually operated on a single large computer sneen that displays both the above menu and the images as they are acquired. Appendix I: Confocal Microseopes 420 Olympus Olympus Corporation he Olympus Corporation pro duces a large range of high quality optical instruments, from digital cameras to T research grade light microscopes, including a number of objective lenses specifically designed for use in the biological sciences. The Olympus laser scanning confocal microscope scan heads, wh ich can be attached to a wide range of upright and inverted microscopes, are capable of excellent resolution and offer great versatility. The latest instrument from Olympus utilises spectral separation as weil as the established technology of dichroic mirrors and optical filters for separating the fluorescent light into various regions of the light spectrum. OLYMPUS CONFOCAL MICROSCOPES Olympus has recently introduced the fully computer controlled spectral separation dual sc an Fluoview 1000 (FVIOOO) confocal microscope, but the previous manually controlled scan head of the Fluoview 300 is still manufactured for laboratories that require high quality optics, but do not necessarily require the added versatility (and greater expense) ofthe computer controlled scan head. Figure A-4l. Olympus Fluoview 1000 Confocal Microscope. Olympus manufactures two laser scanning confocal microscopes for use in the biological seien ces, the fully computer controlled spectral separation Fluoview 1000 (above) and the manually adjusted Fluoview 300 (Figure A-44). The sc an head can be attached di rectly to either an upright or inverted research grade light microscope, and in this case is attached to the rear access port 01' an Olympus inverted microseope. This photograph was kindly provided by Olympus Australia. Olympus Corporation Olympus Europe GmbH Olympus America Ine. Olympus Australia Pty Ltd Address: Shinjuku Monolith, 3-1 Nishi Wendenstrasse 14-18 2 Corporate Center Drive 31 Gilby Rd Shinjuku, 2-ehrome, Shinjuku-ku, D-20097 Hamburg Melville NY 11747-315 7 Mt Waverley VIC 3149 Tokyo 163-0914, Japan Germany USA Australia Phone: +81 333402111 +494023773-0 +1 631 R445000 +61 3 9265 5400 Weh: www.olympus-glubal ..:om /en/gl obal Appendix I: Confoeal Microscopes Olympus 421 Olympus Confocal Microscopes OIympus Confocal Microscopes Olympus Corporation is a major manufacturer of Olympus currently manufacture !wo laser spot scanning con/ocal optically based instruments, including scanners, microscopes (Fluoview 1000 and Fluol'iew 300) that are used cameras, medical instruments and microscopes. extensively in the biological sciences. Olympus also manufacture a Nipkow disk scan head (MZX50-CF) thaI is mainly used as a Olympus manufactured the first Japanese made semiconductor inspection microscope. microscope m 1919 under the name Takachiho Laser Scanning Confocal Microscopes Seisakusho. The company, known as Olympus since 1921, now designs and manufactures a wide range Fluoview 1000 A dual scan confocal microscope with spectral separation capabilities. Two of "student" level and "research grade" spectral separation channels and up to microscopes, including stereomicroscopes, upright three optical filter separation channcls, in microscopes and inverted microscopes. addition to a single transmission detection channe!. Fully computer controlled with a Although Olympus was originally known for their single adjustable confocal pinhole. production of good quality "student" microscopes, Multiple lasers can be attached via over the past few years the quality of the research fibreoptic connections. grade microscopes, and the range of high quality f-F-Iu-o-v-ie-w-5-0-0---1-Th-e-F-lu-o-v-ie-w-50-0-la-s-er-s-c-a-nn-i-ng-c-on-t<-o-ca-I-I obj ective lenses that Olympus produces, have microscope is sold as a confocal increased greatly. microscope suitable for multi-user The "research" grade objective lenses manufactured facilities that offers considerable versatility with tried and true dichroie mirror and by Olympus include oil and water ImmerSIOn optical filter technology. The Fluoview objectives, dipping objectives and non-immersion 500 scan head is fully computer controlled, objectives, many of which are specifically designed with up to four fluorescence channels and for the biological sciences, including specialised one transmission channe!. Multiple lasers can be attachcd via fibreoptic connections. lenses for confocal microscopy. This instrument has now been replaced by Olympus produces two confocal microseopes for the Fluoview 1000. 1------+------1 the biological sciences, the Fluoview 1000, which is Fluoview 300 Designed as a "personal" confocal fully computer controlled and capable of spectral microscope, with a manually operated scan separation, and the Fluoview 300, which has a unit, with excellent optics using manually operated scan head and utilises dichroic conventional dichroic mirrors and optical filters. Two fluorescence channels and one mirrors and optical filters for light separation. The transmission channel are available. A weil established technology of using dichroic single laser can be attached directly to the mirrors and optical filters to both select the laser scan head via a fibreoptic connection, or lines used, and to separate the fluorescence signal, multiple lasers can be used by incorporating the additional lasers into the means that the Fluoview 300 can be produced at Olympus laser combiner. considerably lower cost compared with the "high "------'------' end" confocal microscope instruments from Nipkow Disk Confocal Microscope Olympus and other manufacturers. The manually ,-~------,------~------, MX50-CF Real time confocal microscope based on a operated Fluoview 300 model is still an excellent Nipkow disk scan head and mercury or choice where high quality optics are required but a xenon lamp illumination. Mainly used as a lower purehase price and in some cases the ability semiconductor inspection microseope. to readily modify the various optical components L-______L- ______~ Earlier Confocal Microscope are important. rF-y"'X--,--c:::.::.c-=.:.:.::r-'E"-ar':"ly"""m'-'O-'d-"e-'l-c-o-n-fo-c-al-m-ic-r-os-c-o-pe-f-ro-m-' Olympus also produces a Nipkow disk based Olympus, with either two fluorescent confocal microscope specifically for use in the channels or one fluorescent and one materials sciences. transmission channel. Appendix I: Confocal Microscopes 422 Olympus Components of the Olympus Fluoview 1000 The Olympus Fluoview 1000 laser spot scanning confocal microscope is sold as a visible light instrument. but can be readily configured by the user to incorporate UV or multi-photon microscopy. ~ The Olympus Fluoview 1000 confocal microscope is a fully computer controlled dual scan instrument with two spectral separation channels and an additional !Wo detection channels that utilises dichroie mirrors and optical filters. Saut"-' The Fluoview 1000 scan head is attached to the rear li ght path of either an inverted or an upright Olympus research microseope. Fully automated sc an head (computer controlled) and is relatively large, containing all of the optics, as weil as the scanning mirrors. pinhole and photomultiplier tubes. Spectral channels: Two independent spectral separation channels with separate diffraction gratings. Dichroic mirrors: Mounted inside the scan head are computer-controlled wheels that contain a number of dichroie mirrors used to separate the laser excitation light from the emitted Iluorescence, and also to separate different Iluorescence emi ssions in multi-Iabelling applications. These mirrors are used for the initial separation of different wavelengths for both the spectral separation channels and the optical filter based separation channels. Barrier filters: Computer-controlled wheels of barrier filters are mounted in front of each of the photomultiplier tubes used for channel 3 and 4 (the optical filter separation channels). These barrier filters, in combination with the dichroic mirrors, provide the means by which various fluorescent colours can be separated. Variable size confocal iris: A single common computer controlled variable size confocal iris. Light detectors: Up to four photomultiplier tubes. Scanning mirrors: X & Y fast galvo scan mirrors, zoom, bi-directional & scan rotation capabilities. SIM (Simultaneous scanner): the instrument is capable of simultaneous but independent scanning with !Wo different lasers or laser lines . • Lasers: A variety of lasers can be mounted directly onto the sc an head via fibre optics. However, a more versatile option is to use the Olympus laser combiner, which can be used to combine the output from a number of different lasers. This includcs the Kr/Ar, Ar-ion, HeNe, 405 nm violet and 440 nm blue diode, single line Kr-ion, UV and IR lasers. Laser attenuation: AOTF filters located within the laser combiner unit are used to altenuate the amount oflaser light reaching the sampie (in the range ofO.1 to 100%). Laser line selection: Suitable computer controlled optieal filters for seleeting the appropriate laser line ean be placed in front of the laser beam be fore the laser light enters the fibreoptic cable for transfer to the sean head. Olympus research grade inverted or upright microscopes. Multi-channel fluorcscence, spectral imaging, backscatter (relleetance), transmission. Box size user selectable up to 4096 x 4096 pixels, zoom and panning. Dual scanning, simultaneous and sequential multi-channel seanning. Time lapse imaging. 12-bit data aequisition and processing, saved as 8-bit or 12-bit files. Region of interest sc an (clip scanning), including two independent scts of regions of interest when dual seanning. Rotated scan, line seanning and free-Iine seanning. A single photomultiplier tube transmitted light detector. Bright-field, Phase and Die imaging capabilities, depending on the optics installed in the light microscope. , ...... CGarroI The Fluoview 1000 confoeal microscope is fully computer controlled with no provision for manual adjustment of aequisition parameters. ~ High-end PC computer to control and acquire images. Dual screen computer monitors are used for displaying the control panel and the images as they are collected. '8ft Fluoview software nmning under Windows XP operates the Fluoview 1000 sc an head. Opdom Dual or single scanner, optional 4'" fluorescence detection channel, fibre optie output and non-confoeal detectors. '1odrlI Fluoview 1000 (spectral + filter system, or filter system onl y). the manually operated Fluoview 300 (see page 427) and the previously manufactured Fluoview 500 (see page 424). Appendix I: Confocal Microscapes OIympus 423 FLUOVIEW 1000 CONFOCAL MICROSCOPE The Fluoview 1000 dual scan spectral separation confocal microscope is designed for high resolution imaging in the biological sciences. The scan head is directly attached to a variety of ports on either an upright or inverted research grade light microscope. The Fluoview 1000 scan head (Figure A-42) contains all of the necessary components for scanning the laser be am across the sampIe, the separation of various wavelengths of fluorescent or reflected light and their detection by up to four independent channels. A range of lasers can be connected to the scan head either directly by fiber optic connection, or by using the Olympus laser combiner platform. ~ barrier filters diffradion gratilgs Fluorescent or ref1ected light from ~~===~;;;;;;=====:::J.~======:;':;;'::==lIIF::::::: the microsoope adjustable size confocal dichroie mirrors pinhole Figure A-42. Olympus Fluoview 1000 Scan Head Layout. The Olympus Fluoview 1000 sc an head contains up to four dctection channels. two of which are based on spectral separation using a diffraction grating and two use conventional dichroic mirrors and baITier filters (a more detailed description ofthe method oflight separation used in the Olympus Fluoview 1000 scan head is given in Figure 3-13 on page 84). A single variable size confocal pinhole or iris is used for a11 detection channels. Thc lasers are mounted on a separate laser combincr and attached to the sc an head via separate fibreoptic connections. The scan head is attached to the rear light entry port on an inverted microscope (as shown in Figure A-4I) or the top illumination port on an upright microseope. This figure is adapted from a diagram kindly provided by Olympus Australia. Appendix I: Confucal Microscopes 424 Olympus The scan head contains Up to four independent detection channels (Figure A-42). The two spectral separation detection channels contain diffraction gratings and variable width slits that can be moved to select specified regions of the light spectrum. The slits can be used to specify specific wavelength ranges that are to be directed to each channel, or they can be used to collect a very narrow band oflight (down to I nm) for spectral imaging. The various spectral separation techniques used by different confocal microscope manufacturers are described in detail on page 81 - 85 in Chapter 3 "Confocal Microscopy Hardware". The scan head also contains one, or if required, two "conventional" detection channels that utilize dichroic mirrors and optical filters to separate various regions of the light spectrum. The spectral separation and the optical filter based channels can be used to simultaneously image up to four different fluorophores. Altematively, the spectral imaging channels can be used to collect spectral information from fluorophores with highly overlapping fluorescence emission spectra. Linear unmixing is then used to separate the contribution from the individual fluorophores, which is then displayed as separate images. A single computer controlled variable size confocal iris or pinhole is used to select only the focal plane within the specimen. Non-confocal detectors (positioned within the scan head prior to the confocal pinhole) for multi• photon microscopy are also available as a optional accessory. Feedback control of laser intensity, high-sensitivity photomultiplier tubes with photon counting capability and the ability to collect spectral information over a broad range (400-790 nm) at high resolution make this instrument a highly versatile microscope for biology, and particularly for live cell imaging. SIM (Simultaneous) Scanner The Fluoview 1000 scan head is capable of simultaneous and independent dual scanning with two different laser lines or lasers. This feature allows one to perform real-time fluorescence photobleaching (FRAP) and uncaging experiments by using one laser to uncage or bleach a specified region ofthe specimen while a second laser is used to collect the image. This feature also allows the simultaneous collection of two independent sets of regions of interest (ROI) within the sampie. FLUOVIEW 500 CONFOCAL MICROSCOPE The Fluoview 500 confocal microscope has a fully automated scan head with a fibreoptic connected computer controlled laser platform. The instrument is capable of imaging up to four fluorescence channels and a single transmission channel simultaneously. Although the instrument is fully computer controlled the relatively simple optics do mean that the scan head can be readily customised by more experienced users. This includes changing the dichroic mirrors and optical filters and the attachment of pulsed infrared multi-photon lasers. Fluoview 500 Scan Head The Fluoview 500 scan head shown in Figure A-43 is mounted directly onto the side of an Olympus inverted microscope, but can also be mounted in a number of other positions, including to the rear of the microscope as shown in Figure A-4l. The separation of the fluorescent light into defined regions of the light spectrum is achieved by using the established technology of optical filters and dichroic mirrors (Figure A-43). The scan head contains computer controlled variable size pinholes for each of up to four detection channels. This is in contrast to the single pinhole (with a choice of sizes) used for both channels in the Fluoview 300 scan head, and the single variable size pinhole used in the Fluoview 1000 scan head. Custom designed optical filters and dichroic mirrors can be readily fitted into the filter wheels used in the Fluoview 500 scan head. A number of different lasers can be directly fibreoptic connected to the scan head, or they can be mounted on a separate laser platform which contains laser line selection and attenuation filters. Pulsed infrared lasers for multi• photon microscopy can be readily attached directly to the Fluoview 500 scan head. Galvanometric scan mirrors can be rotated to sc an any dcfined area and box size within the microscope field of view. Although several defined regions of interest (RO!) can be scanned simultaneously, this instrument is not capable of dual scanning (where two completely independent ROI can be scanned simultaneously). Appendix I: Confocal Microscapes Olympus 425 Components of the Olympus Fluoview 300 and 500 The Olympus Fluoview 300 and 500 laser spot scanning confocal microseopes are sold as visible light instruments, but can be readily configured by the user to incorporate UV or multi-photon microscopy. ~ The Olympus Fluoview 300 confocal microscope is a manually operated instrument that is affordable o for individuallaboratories. The previously manufactured Fluoview 500 is fully computer controlled, but has now been replaced by the Fluoview 1000. The Fluoview 300 and 500 instruments are based on o the use of dichroie mirrors and optical filters. Sra lIMd The Fluoview scan head is attached to either an inverted or an upright Olympus research microseope. The attachment locations include the familiar top position for an upright microscope and the side ~ position for an inverted microscope, or the rear light path of either an upright or inverted microseope. Fluoview 500 Fully automated scan head (computer controlled) and is relatively large, containing all ofthe optics, as weil as the scanning mirrors, pinholes and photomultiplier tubes. Dichroic mirrors: Mounted inside the scan head are computer-controlled wheels that contain a number of dichroie mirrors used to separate the laser excitation light from the emitted fluorescence, o and also to separate different fluorescence emissions in multi-Iabelling applications. Barrier filters: Computer-controlled wheels of barrier filters are mounted in front of each of the o photomultiplier tubes. These barrier filters, in combination with the dichroie mirrors, provide the means by which various fluorescent colours can bc separated. lr) Variable size coufocal iris: A computer controlled variable size "pinhole" is mounted in front of each photomultiplier tube within the scan head. Light detectors: Up to four photomultiplier tubes. Scanning mirrors: X & Y fast galvo scan mirrors, zoom, bi-directional & scan rotation capabilities. Fluoview 300 Manually operated dichroie mirrors, optical filters and pinhole size adjustment within the scan head. Dichroic mirrors: Various dicltroic mirrors are mounted on manual sliders within the scan head. Barrier filters: Manually adjusted sliders are used to change the barrier filters within the scan head. These barrier filters, in combination with the dichroie mirrors, provide the means by which various fluorescent colours can be separated. Confocal iris: Fluoview 300 scan head has a manually operated five-position pinhole unit Light detectors: Two photomultiplier tubes. Scanning mirrors: X & Y fast galvo scan mirrors, zoom, bi-directional & scan rotation capabilities . • L.--. Lasers: A variety of lasers can be mounted directly onto the scan head (tltree attachment ports for the Fluoview 500 and one for the Fluoview 300). However, the Olympus laser combiner, can be used to combine the output from a number of different lasers. This includes the Kr! Ar, Ar-ion, HeNe, 405 nm violet and 440 nm blue diode, single line Kr-ion, UV and IR lasers. Laser attenuation: Neutral density filters or AOTF filters located within the scan head or laser combiner unit, are used to attenuate the amount oflaser light reaching the sampIe. Laser line selection: Suitable optical filters for selecting the appropriate laser line can be placed in front of the laser beam before the laser light enters the fibreoptic cable for transfer to the scan head. These filter wheels are controlled manually in die Fluoview 300 and by computer in the Fluoview 500 , 1II:r..:upe Olympus research grade inverted or upright microscopes. Multi-channel fluorescence, backscatter (reflectance), transmission. Box size user selectable up to 1024 x 1024 pixels, zoom and panning. Simultaneous and sequential multi-channel scanning. Time lapse imaging. 12-bit data acquisition and processing, saved as 8-bit or 12-bit files. Single region of interest scan (clip scanning), rotated scan, line scanning and free-line scanning. 1'1~ A single transmission photo diode transmission detector. Bright-field, Phase and DIC imaging capabilities, depending on the optics installed in thc light microseope . .\ __ CoaIroI Thc Fluoview 500 confocal microscope is fully computer controlled with no provision for manual adjustment of acquisition parameters. The Fluoview 300 is a manually operated confocal microseope. CompuMr High-end PC computer to control and acquire images in the Fluoview 500 confocal microseope. The 1-_--''-'- Fluoview 300 scan head is a manually operated instrument, but uses a computer for image acquisition. A single large computer screen is used for both instruments. SoIwwI: Fluoview software running under Windows NT/2000 operates both the Fluoview 500 and Fluoview 300 sc an heads (with the appropriate components loaded during installation). todds Fluoview 300 (manually controlled), the previously manufactured Fluoview 500 (computer controlled) and the spectral separation Fluoview 1000 (see page 423). Appendix I: Confocal Microseopes 426 Olympus Dichroic mirrors fOf ftuorescence separation ~ Dichroie mirrors for laser (ine selection ~ Fibre oplic connections fOf up 10 Ihree lasers lines (each (ine can have mOfe Ihan one laser by ...... x-V galvanometer using a laser combiner unil) scanning mlrrors Direct connection to lhe microscope Figure A-43. Olympus Fluoview 500 Scan Head. The Olympus Fluoview 500 scan hcad contains most of the optical components neccssary for confocal microscopy. This includes the dichroie mirrors. optical filter wheels. scanning mirrors. variable size pinholes. photomultiplier tubes (up to four) and laser line selector. However. the lasers are mounted on a separate laser combiner and attaehed to the se an head via three separate tihreoptic connections. The scan head is shown here attached to the side port of an Olympus inverted microseope. hut ean also he attached to the rear light entry port on an inverted mieroscope (Figure A-41) or the top video port on an upright microscope. This figure is adapted tram a diagram kindly provided hy Olympus Australia. Appendix I: Confoeal Mieroscopes Olympus 427 FLUOVIEW 300 CONFOCAL MICROSCOPE The Fluoview 300 confocal microscope is a manually operated confocal microscope with somewhat limited versatility but with excellent optics based on dichroic mirrors and optical filters for separating the fluarescent light. Severallasers can be connected via fibreoptic connections. Fluoview 300 Scan Head The Fluoview 300 scan head, shown here attached to an unusual upright microscope that has a rigidly fixed microscope stage (Figure A-44), is manually operated. Simple sliders are used to position the correct dichroic mirror and optical filter far imaging various fluorophores. The manual settings are very simple to adjust, with a graphical computer display of user settings for specific dye combinations. There is a choice of 5 different sizes for the single confocal pinhole (in contrast to the variable size pinhole for each channel in the Fluoview 500 instrument). The Fluoview 300 confocal microscope scan head is significantly less expensive compared to many other confocal microscopes, but with excellent optics, and the added advantage that the scan head can be readily customised for individual use. This inc1udes using customised dichroic mirrors and optical filters, as weil as the direct attachment ofpulsed infrared multi-photon lasers. Fluoview 300 scan head Manual sliders for dichroic and optical filter adjustment Five position controI knob for changing the confocal IriS size "Fixed stage for live celi l tissue studies "x-Y position controls mave the whole microscope - including Ihe altached Fluoview 300 scan head Figure A-44. Olympus Fluoview 300 Scan Head Attached to a BX61Wl Fixed Stage Microscope. The Fluovie\\' 300 manually operated ronfoeal microscope sc an head is shown here attached to the unusual Olympus designed fixed stage upright microseope. The scan head has been mounted on thc top camera port on the microscope, but has been eonveniently positioned to the rear of the microscope using a simple mirror junetion box (whieh also allows for the attaehment of a CCD eamera in this example). The stage on this microseope, as the name suggests, is fixed to the bench - with all movement and foeus eontrols being aehieved by moving the microseope. This photograph was kindly provided by Olympus Australia. Appendix 1: Confoeal Microscopes 428 Olympus Fluoview Software The Fluoview software provided by Olympus is designed to operate the Fluoview 300, Fluoview 500 and the Fluoview 1000 scan heads. During installation of the software the various components necessary for each of the scan heads is installed. The following figures are designed to give you abrief overview of the main menus for operating the Olympus confocal microseope, based on the display used for the Fluoview 500 instrument. For a more detailed discussion you should refer to the Olympus confocal microscope operating manual. Image Collection Panel The main display screen (Figure A-45) contains the main control panel (described in more detail in Figure A-46) on the left hand side of the active "live" image display. Aseries of "tabs" on the top of the display screen can be used to access a number of recently imaged or previously opened image files. Acquisition control panel active ive display tab active Figure A-45. OIympus Fluoview Software Interface. The Fluoview 500 and 300 eonfoeal mierascopes are both operated by the Fluovicw software, ",hieh is customised tor installation on each of these instruments. All of the main contral features are shown in the panel on thc left. Two images are shown in this particular display, with the top tab on the sereen allowing you to display at the click of amause a number of previous experiments without thc need to go looking for the file. Detailed information on the contral panel on the left hand side ofthis screen display is given in Figure A-46. Appendix I: Confoeal Mieroseopes Olympus 429 Main Control Panel Fast scanning for positioning Ac uire tab active -~~~~-~_- sampIe & focussing (lass lines in scan) Third & fourth channel not selected I .... I-:::--~- Controls tor active c:hannet Mouse dick here to display (c:hannel 2) channel one controls ui~L2I~~~----t- POJHlP button for adjusting laser intensity & confocaJ iris slze Irnaging mode ~ r. XV XYT X'I'ZT r. SWf_ XV-Norm Normal ; regu!a' '~y scan Fa.t =bid_1 sc;ennlng - scan • smaIer bei< slze I I~ 0 ~ I;>J Clip " Zoomln =scan a smaI box oIze roIaIed 10 I nMI Fm Clip ZoomIn Seq any angle (" Llne JeT-Norm Seq = scan """"fMIIs __tiaIIy ( , (" Depth XZ one tina Of one """'" 9 1 a lime) (" P.... Box coIlection size On pixels) lens_ in use"""""'-' (..-..._- - • g lXII. BX8-1) __...... ~ ~~~~~~~~~~~~~_ Rotate scan Move scan area around Confocal scan zoom within fiek:t 01 view - --...... - location ot scan within !leid of view Change scan speed (three positions onty) Controls for setting the z· steps Flgun 1\46. Olympu Fluovlew Co.rrol P•• d. SI"" S_ Tbc FIuoview conlJOI panel h all Ihc 13m and I ..1 .... btI contral that you will nccd f, r 01ltt""" thc 015p,", ! Appendix 1: ConfocaI Microseopes 430 Olympus Instrument Settings Tabs Image coUectlon s tt ngs ... _- / ~ r-: - -- ~ -==--J FIIaI. w'" I r. X2 X4 Aftqglng fl .... 10 _n.. -_ " "wl ln ehe mag. _l .... fHl ---- 1----- Z5 .. _ IX z ..tage focus Time course settings control tab actlve IZI ---,,----. umlt, i I 'lfte" ·Xl - IE!I .e<: Instrument settings for varlous_.. dye - comblnatJons Figure A-47. Olympus Fluoview Instrument Settings Tabs. On the lower portion of the main contral panel (shown in Figure A-46) are aseries of tabs far displaying submenus for chan ging various settings on the microseope. Appendix I: Confocal Microscopes Atto Bioscience 431 Atto Bioscience tto Bioscience produces a number of confocal microscopes based on the spinning Nipkow disk technology. AThese instruments are particularly suited to live cell imaging due to the high acquisition speed and relatively low excitation light intensity required. The Nipkow spinning disk scan head produced by Atto Bioscience, called CARV, is sold as an independent unit that can be attached to a new or existing laboratory microscope (Figure A-48), or as an integrated unit, inciuding lamp source, CCD camera and associated software from both Atto Bioscience and a number of other vendors. ATTO BIOSCIENCE CONFOCAL MICROSCOPES Atto Bioscience produces the CARV confocal microscope scan head (Figure A-48) as an independent unit that can be attached to a variety of upright or inverted microscopes. Atto Bioscience also produces the Pathway HT confocal microscope (based on the technology used in the CARV scan head), that is a fully integrated instrument designed for high-throughput screening of living cells. Figure A-48. Atto Bioscience CARV Nipkow Disk Confocal Microscope. The CARV sean head (shown on the left) ean be attaehed to either an inverted or upright mieroseope from a variety ofmanufaeturers. The image ean be viewed by looking direet1y down the eyepieees on the sean head, or a CCD eamera (shown here attaehed to the top e-mount port on the sean head) ean be used to eapture the image. This instrument is eapable of high-speed imaging (up to 200 frames per seeond, depending on the speed and sensitivity ofthe CCD eamera attaehed). This photograph was kindly provided by Baggi Somasundaram, Atto Bioseienee, USA. Atto Bioscience web: www.atto.coml Address: 15010 Broschart Road Rockville, MD 20850, USA Phone: -I (301) 340 7320 I FAX: I +1 (301) 340 9775 Appendix I: Confoeal Mieroseopes 432 Atto Bioscience Atto Bioscience Confocal Microscopes The Atto Bioscience CARV Nipkow disk Nipkow Disk Confocal Microscopes confocal microscope sc an head was The confocal microscopes produced by A/lo Bioscience utilise a originally marketed by Atto Instruments, an Nipkow disk with a an array of 20,000 pinholes. The image can be optical instrumentation company based in viewed directly by looking down the viewing port on the scon head, or a CCD camera can be a/lached to capture the image. the USA, through an exclusive relationship The instruments are designed for live cell imaging, and are with Carl Zeiss, Germany. The CARV particularly suited to either fast cel/ular [Iuxes or relatively lang time instrument, and more recently the Pathway series. HT confocal microscope, are now produced and marketed directly by Atto Bioscience. CARV Single channel Nipkow disk confocal microscope scan head that attaches to a range of The CARV confocal microscope is commercially available light microscopes. This marketed by Atto Biosciences and through includes upright and inverted microscopes. The a number ofworld-wide distributors. image can be viewed directly through the viewing port on the scan head, or a high-sensitivity CCD The Atto Bioscience confocal microscopes, camera can be used to capture the images. being based on the Nipkow disk, are The instrument is capable of very high speed particularly suited to live cell imaging. imaging (the Nipkow disk is scanning at 200 frames per second, but the actual speed of These instruments are significantly faster imaging is determined by the sensitivity and than the laser spot scanning confocal speed ofthe attached CCD camera). microscopes, and due to the large number The relatively inexpensive CARV scan head is of fine points of light that are scanned sold as an independent unit, but you will need to across the sampie the light intensity at any purchase a CCD camera and suitable software to capture images. one point is relatively low. The Atto Bioscience confocal microscopes Pathway HT High-throughput automated confocal imaging instrument based on the CARV Nipkow disk utilise conventional mercury or xenon lamp teehnology. Designed to handle 96 or 384 well irradiation to excite the fluorophores. This eulture plates, or conventional microseope slides. has the advantage that one can choose Automated software provides for the colleetion of wavelengths of excitation from UV through images from a large number of sampies, and the ability to analyse eaeh individual eell within a to infrared, and the existing lamp using for large number of images for multi-parameter conventional epi-florescence microscopy response to various stimuli. can be attached to the scan head when This instrument is sold as a fully integrated unit fitting the instrument to an existing research and is thus significantly more expensive than the microscope. CARV scan head alone. CARV CONFOCAL MICROSCOPES The CARV confocal microscopes are instruments based on a scan head that incorporates a single Nipkow disk spinning at high-speed. These instruments are capable of scanning at significantly higher speeds compared to a laser spot scanning confocal microscope. Full frame image collection can routinely be obtained at several frames per second (up to 100 fps, depending on the CCD and light levels), compared to approximately 1 fps for a laser spot scanning system. The CARV scan head can be purchased as a separate independent unit that can be attached to a light microscope. In this case you will then need to attach a light source, a CCD camera, and install suitable software for image acquisition. However, a number of companies put together an integrated unit that includes the microscope, a CCD camera and image collection software. A fully integrated Nipkow disk confocal microscope, the Pathway HT, is designed as a high-throughput instrument for screening large numbers of cells. This instrument is bascd on the CARV single Nipkow disk scan head, with the added versatility of dedicated software to collect and analyse images from a large number of sampies. Appendix I: Confocal Microscopes Atto Bioscience 433 Components of the CARV Confocal Microscope A real time Nipkow seanning disk eonfoeal mieroseope manufaetured by Atto Bioscience. The relatively small and compaet CARV sc an head ean be purehased as aseparate unit that ean be attached to an existing research microseope. Altematively you ean purehase a fully funetional confoeal microscope, which inc1udes the sean head, the microseope, the CCD eamera and operating software. L-___..; ~= High-speed Nipkow spinning disk confoeal mieroseope designed around the CARV scan head. Illumination by using a mercury or xenon arc lamp. The image ean be direetly viewed through the scan head eyepieees, or the image ean be aequired using a CCD camera. The scan head is relatively inexpensive compared to other eonfoeal microseopes, but the total eost of the system will be determined by the price of the attached CCD eamera (high-speed eooled CCD eameras are very expensive), and the type of software required (no software is provided). A simple sliding lever allows one to readily switch between eonfoeal and non-eonfoeal viewing. Und CARV Nipkow disk unit eontains a single Nipkow disk (without the miero-Iens array disk), dichroic mirrors and optieal filters for separation of various regions of the light speetrum. The relatively small robust scan head is faetory aligned. A mercmy or xenon arc lamp is attaehed to the side of thc scan head. Fixed "pinhole" Nipkow disk: The pinhole disk contains 20,000 pinholes (at any one time about 1000 pinholes sean the sampie) that rejeet the out-of-focus light. Due to the design of this instrument the size ofthe pinholes eannot be adjusted. Diehroic mirrors and optical fIlters: Mounted within the CARV sean head are eonventional (the same as those used in the Zeiss wide-field epi-fluoreseenee microseope) dichroie mirror / optieal filter blocks, which ean be ehanged depending on the light separation required. A three position turret for choiee of optical filters is provided. CD The disk sean rate is at 200 frames per seeond, although the speed at which the CARV confoeal mieroscope can operate is detennined by the sensitivity and speed of the CCD eamera that is attached to the sc an head. A highly sensitive and high-speed CCD eamera will be required for low light fluorescence imaging applications and can readily aehieve speeds of 50 fps at 512 x 512 pixels. I.J&hIlIoIIIu Mercury or xenon arc lamp: A wide range of exeitation wavelengths, from 340nm UV through to near IR, are available, depending on the filter block used. The mereury or xenon lamp housing on a eonventional wide-field epi-fluoreseenee mieroseope can be transferred to the CARV sean head when retrofitting the scan head to an existing mieroscope. Exeitation wavelengths are seleeted by the use of speeifie optieal filters. The CARV scan head can be attached to a number of upright or inverted microseopes, including easy attaehment to existing laboratory mieroseopes. High-speed single ehannel fluoreseenee eonfoeal or baekseatter (refleetanee) eonfoeal imaging. Easily switched to bright-field / DIC imaging and wide-field epi-fluoreseence imaging. Direet viewing ofthe eonfoeal image is possible. Very high-speed imaging (up to 100 fps, depending on the speed ofthe attaehed eamera). Multi-fluoreseence imaging ean be aehieved by sequential seanning and adjustment of optieal filter positions. Zoom, ROI, panning ete are not possible due to the way in whieh the image is eolleeted via a CCD eamera. Excitation / emission filters ean be fitted. n...... Transmission images can be obtained by moving a simple slider in the sean head. o M.ullCoaii'oI The sean head is manually operated, with the computer being used to eapture images from the CCD. ~ The instrument is manually operated and the image can be viewed direetly by looking through the eyepieees on the sean head. Image eapture is via a CCD camera, using eommercially available software. ~ Commereially available software for eapturing images from the CCD eamera is used to eolleet digital images . ....____ ~_ IodfII~ CARV: The CARV sean head ean be readily adapted to a variety ofmieroseopes. Pathway HT: A high-throughput automated eonfoeal mieroseope based on the C AR V sc an head. Appendix I: Confoeal Microseopes 434 Atto Bioscience CARV Scan Head The CARV confocal microseope scan head is a finely engineered, robust, factory aligned independent instrument that can be readily attached to a conventionallight microscope (see Figure A-48). The scan head contains the single Nipkow disk (Figure A-49), wh ich is spinning at adjustable speeds of up to 200 frames per second, although the ac tu al image collection speed will normally be significantly lower than this (15 to 25 frames per second), and is highly dependent on the speed and sensitivity of the camera and the amount of fluoreseenee in the sampie. A eonventional mercury or xenon arc lamp is attaehed to the se an head, providing exeitation light from UV through to IR - depending on the partieular dichroie mirrors and optieal filters used. Aseries of conventional wide-field epi-fluoreseence mieroscopy optical filter blocks are used to separate out the various regions of the light spectrum and to direct this light to either the binocular viewing port or to an attached high-resolution CCD camera. A wheel containing up to three user selectable optical filter blocks is mounted in the scan head. Further optical filter blocks can be swapped for those already in the filter wheel. A z-stepper, when installed on the microscope can be used to colleet optical sections for 3D reconstruction. The ability to collect images at relatively high-speed means that a full 3D optical stack can be collected in seeonds or less - allowing one to collect almost real time 4D data sets (3D + time). Sophisticated 4D or time lapse imaging is dependent on having suitable software installed to control the CCD camera acquisition time and z-stepper. CARV scan head Sampie Nlpkowdlsk and slider 100% mlrror Microscope Mereury or xenon are lamp HIgh-resolution eooled CCD eamera Blnocular vlewlng port Figure A-49. Atto Bioscience CARV Nipkow Disk Scan "ead. The light path for the CARV scan head is shown diagrammatically. The light is focussed through the large array of pinholes on the Nipkow disk (see page 91), resulting in a large number of finely foclIssed spots scanning the sampIe at a rate of 200 frames per seeond. Illumination is provided by a conventional mcrcury or xenon are lamp attached to th e sean head. The image can be viewed directly by looking through the binocular viewing port on the scan head. A CCD camera can be attached to the sean head to eapture the image. Exeitation and emiss ion filters ean be ehanged by using a different filter cube (the conventional wide-field epi-fluoreseence filter blocks used in Zeiss research grade light microscopes) .. Appendix I: Confocal Microscopes Yokogawa 435 Yokogawa Electric Corporation he Yokogawa CSUIO and CSU21 spinning Nipkow disk scan heads are produced in Japan by the Yokogawa T Electric Corporation. The design of these scan heads has resulted in an instrument that is unusually fast for a laser-scanning microscope (routinely 10 to 20 frames per second, but can be as high as 100 or more frames per second). Furthermore, the multi-point scanning technique used in this instrument, means that the light intensity at each individual point of light is much less intense compared to a conventional single point scanning confocal microscope - resulting in greatly decreased photo bleaching of the sampIe. The Y okogawa scan heads are assembled into confocal microscopes by a number of manufacturers, including PerkinEImer, and VisiTech International, which are discussed in so me detail on the following pages. The principles involved in confocal image acquisition in the Yokogawa Nipkow disk / micro-Iens array scan head are described in more detail in on page 91. Further details on the Y okogawa scan head can be found in Methods in Cell Biology Vol. 70 edited by Brian Matsumoto, chapter 5 "Direct-View High-Speed Confocal Scanner - the CSU 10" by Shinya Inoue and Ted Inoue (see page 349 in Chapter 15 "Further Reading" for more details). CSU10 SCAN HEAD The compact and precisely engineered Nipkow disk CSUIO scan head (Figure A-50) is designed and manufactured as an Figure A-50. Yokogawa CSUlO Sc an Head independent unit. The scan head contains a The CSU 10 micro-Iens array Nipkow disk scan head is produced in dual spinning disk - where the first disk is an Japan by Yokogawa, and is assembled into complete confocal microscope systems by a number of companies. The scan head can be array of micro-Ienses that are accurately attached to either upright or inverted microscopes from a variety of aligned with a corresponding array of manufacturers. The instrument is capable of very high-speed confocal "pinholes" on a second disk (see diagram in imaging - the final image acquisition speed achieved depends on the Figure A-51, and Figure 3-18 on page 92). speed and sensitivity of the attached CCD camera. This image was Light passing through each micro-Iens is kindly provided by George L. Kumar (PerkinEImer. USA). directed through a dichroic mirror and Yokogawa Electric Corporation Web: www.yokogawa.com! Email: csuCacsv.yokogawa co.ip .\ddre'!s Confocal Scanner Business Group BIO Centre, ATE Business Division Y okogawa Electric Corpora ti on 2-9-31 Nakacho, Musashinoshi, Tokyo, 180-8750, Japan Phon" +81 442 52 5550 I FAX, I +81 442 52 7300 Appendix I: Confocal Microscopes 436 Y okogawa focussed onto the "pinhole" of the second Nipkow disko The technique of focusing the light through each individual pinhole greatly increases the light efficiency of this instrument compared to those using a single Nipkow disko The light from each of the individual pinholes then passes through the objective lens of the microscope - resulting in thousands of points of light being projected onto the sampie. The retuming fluorescent light is directed back through the pinholes on the first disk (rejecting any out-of-focus light) and reflected by the dichroic mirror (a "reverse" dichroic mirror compared to the point scanning confocal microscopes) onto a CCD camera. The micro-Iens array disk and the Nipkow disk are physically attached to each other to eliminate any problems of Micro-Iens array To the microscope Dichroie mirror -+-~.'~ Figure A-S1. Optical Path ofYokogawa CSUlO Scan Head. The Yokogawa CSUIO sean head eontains a Nipkow disk and a miero-Iens array disk (deseribed in detail on page 93) that are spun at high• speed together to illuminate the sampie with a large number of light points. The eonfoeal image can be viewed live by looking down the viewing port on the sean head, or images ean be eaptured by using an attached CCD camera. Some instruments provide two CCD cameras for simultaneous dual channel imaging. Irradiation is provided by a variety of lasers. This diagram is modified from a diagram kindly provided by George L. Kumar (PerkinEImer, USA). The layout of the Nipkow disk I miero lens array disk in the Yokogawa sean head is shown diagrammatieally in Figure 3-18 on page 92. alignment. The dual disk system is spun at 1,800 rpm (30 rps), which results in 1200 pinholes scanning the sampie at any particular instant. The disks are spinning at 12 times the video rate of30 fps (i.e. 360 fps), which gives 12 frame averages for each video frame. Further enhancement can be achieved by on chip averaging. The 20,000 pinholes in the Nipkow disk have a diameter of 50flm (250 flm apart) to give a light throughput of only 4%. However, by focusing the light through the pinholes using the micro lenses the light throughput of the disk is significantly improved to approximately 40%. The holes are arranged in a "constant-pitch helical pattern" to create even illumination across the field without creating scanning artefacts. The CSU 10 scan head is connected to the camera port of a conventional light microscope (Figure A-51). The confocal image can be observed by direct viewing through the eyepiece (viewing port) on the sc an head, or by simply moving a slider so that the light can be directed to an attached CCD camera. The CSU 10 scan head is made up into fully integrated confocal microseopes by a number of manufacturers (see the following pages). This includes providing a computer with appropriate software for acquiring images and Appendix I: Confocal Microseopes Y okogawa 437 controlling laser intensity and line selection. A grey-scale or three-colour CCD camera, or sometimes two grey-scale CCD cameras are attached for simultaneous dual channel image capturing. Nipkow disk based confocal microseopes do have a number of irnportant advantages compared to the laser spot scanning instruments, but there are also a number of limitations inherent in the technology (discussed in detail on page 91-93 in Chapter 3 "Confocal Microscopy Hardware"). An important advantage of the Nipkow disk based instruments is the ability to do relatively high-speed imaging. Although the CSUIO scan head Nipkow disk is spinning at 360 frarnes per second, the actual speed of image acquisition achieved is considerably less and is highly dependent on the type of CCD camera attached. However, even with a relatively modest CCD camera speeds of up to 20 frames per second can be achieved. This is in contrast to a typical acquisition speed of only one frame per second for a spot scanning system. CSU21 SCAN HEAD The CSU21 scan head (see Table A-I below), also produced by Yokogawa, is similar in design to the CSUIO scan head but does have a number of features that make it particularly suited to very high-speed imaging. The CSU21 scan head has a variable scan speed from 1,800 rpm to as high as 5,000 rpm, which results in a frames scan rate as high as 1,000 frarnes per second. However, the actual image capture rate is still dependent on the sensitivity and speed of the attached CCD camera. The CSU21 scan head is exclusively distributed outside of Japan by PerkinEImer. Table A-l. Comparison ofthe Yokogawa CSUIO and CSU21 Scan Head. Yokogawa produces two Nipkow disk / micro-lens aITay disk sc an heads, the CSU 10 and the CSU21. The CSU2l scan head is significantly faster than the CSU 10, has motorised optical filter changes, can have two CCD cameras attached and has reduced noise levels. A number of manufacturers (discussed on the following pages) produce instruments with modified vers ions of the CSUIO sc an head that may have some ofthe features ofthe CSU21 scan head. CSUI0 scaD head CSU21 scaD head Nipkow disk 20,000 holes (50"m) 20,000 holes (50,lm) Micro-Iens array disk 20,0001enses 20,000 lenses Disk rotation speed Fixed 1,800 rpm Variable 1,800 - 5.000 rpm Eye viewing Yes Yes CCDcamera Single grey-scale or three-colour CCD camera, Single camera or three-colour CCD, with with modification two cameras can be attached. modification two cameras can be attached. ND and excitation filters Manual change Motorized Lasers Various: Fibreoptic coupled Various: Fibreoptic coupled Frame scan speed 360 frames per second As high as 1,000 trames per second (image capture speed depends on CCD camera and light level) Installation C-mount on microscope C-mount on microscope Emission filters Manual filter change Motorised filter change PC or front panel control. Synchronisation NTSC & P AL or slawer speeds NTSC. PAL and synchronisation with various exposure times of CCD cameras. Appendix I: Confocal Microseopes 438 PerkinEImer PerkinEImer Life Sciences he Nipkow spinning disk confocal microscope produced by PerkinEImer is based on the CSUIO micro-Iens T array Nipkow disk scanning head produced by Y okogawa (page 435). The design of this confocal microscope has resulted in an instrument that is unusually fast for a laser-scanning confocal microscope (routinely 10 to 20 frames per second, but can be higher than 100 frames per second). The multi-point scanning technique used in this microscope means that the light intensity at each individual point of light is much Icss than with a conventional single point scanning confocal microscope - resulting in greatly decreased photo bleaching ofthe sampie. PERKINELMER CONFOCAL MICROSCOPES The principles involved in the Nipkow disk confocal microscope are described in more detail on pages 9 I -93 in Chapter 3 "Confocal Microscopy Hardware", and the Yokogawa CSUIO scan head used in the PerkinEImer instruments is described on page 435 ofthis appendix. Inverted light microscope / CCD camera Fibre optic coupling Figure A-52. PerkinEImer Ultra VIEW Confocal Microscope. The PerkinEImer (Wallac) Nipkow disk confoeal microscope is designed around the Yokogawa micro-Jens Nipkow disk scan head (CSUIO). Unlike the point-scanning eonfoeal microscopes, this instrument creates an image by using a CCO camera - and you can directly view the image by using the eyepiece direetly mounted on the sean head. The instrument is capable 01' very high-speed eonfoeal imaging - but the speed aehieved does depend on the type ofCCO eamera attaehed. This image was kindly provided by George L. Kumar, PerkinEImer, USA. PerkinEImer Lire Sciences Address PerkinEImer Life Seienees PerkinEImer Life Seienees PerkinEImer Life Seienees 549 Albany Street Imperiastraat 8, P.O. Box 600 Boston, MA B-1930 Zaventem Knoxfield MOC VIC 3178 USA Belgium Australia Phone 18005512121 ~3227177911 +61392128500 FAX , +61392128595 Appendix I: ConfoeaI Microseopes PerkinEImer 439 Nipkow disk based confocal microscopes are particularly suited to live cells imaging where either high speed imaging of cellular events is required (for example, when imaging fast changes in calcium fluxes) or minimal laser damage is required during long term time lapse imaging (for example, when imaging developing embryos). The instrument can still be used for more conventional imaging of fixed sampies such as immunolabelling, although there are some limitations when compared to a single point scanning confocal microscope (see pages 91- 93 In Chapter 3 "Confocal Microscopy Hardware"). Ultra V/EW Le1/ayaut The Ultra VIEW LCI confocal microscope is sold as an integrated unit, consisting ofthe Yokogawa scan head, a single CCD camera, a microscope, and with computer and proprietary software to control laser line selection and attenuation, scan head, and image acquisition. The Yokogawa scan head is a small and compact instrument that takes up a relatively small area on the microscope bench (Figure A-53). The scan head can be attached to any camera port on an upright or inverted microscope, although the preference would be to install this type of scan head on an inverted microscope due to the significant advantages for using this instrument for imaging live cell or tissue preparations. The PerkinEImer Nipkow disk confocal microscopes are provided with z-focus control using a stepp er motor, or for more accurate and significantly faster z-sectioning high-speed piezoelectric focusing is used. In this way fast 3D data sets can be readily collected to follow cellular functions in 3D over time (4D imaging). Various lasers are readily accommodated by the Ultra VIEW LCI confocal microscope by attachment to the scan head via a fibreoptic connection. The laser does have the advantage of having very precisely defined wavelengths that can be used for excitation, but there is the disadvantage that you will be limited to the excitation wavelength of the lasers you have on hand. Nipkow disk confocal microscopes that utilise the much cheaper and the broad excitation wavelengths of a mercury or xenon arc lamp, are available. Automated optical filter control is available on the Ultra VIEW LC I to allow fast switching of excitation and emission wavelengths for multiple labelling and ratiometric labelling applications. Inverted light mlcroscope Figure A-53. PerkinEImer UItra VIEW Sc an Head. Au enlarged view of the Yokogawa scan head and attached CCD camera as assembled by PerkinEImer Life Sciences. Tbe image can be directly viewed by looking down the "viewing port" while the scanner is operating. The CCD Camera can be of a variety of designs; a high-speed cooled CCD camera is required for low light level high·speed imaging as found in live cell confocal microscopy. A dichroic mirror (located between the micro-Iens array disk and the pinhole array diskl. is used to direct the returning fluorescent light to the CCD camera. A variety oflasers can be attached via the fibreoptic connection. This photograph was taken at the European Molecular Biology Laboratories (EMBLl, Heidelberg, Germany. Appendix I: Confocal Microseopes 440 PerkinEImer Components 0' the PerkinEImer Ultra VIEW The PerkinEImer Ultra VIEW Nipkow scanning disk confocal microscope is assembled using the Yokogawa micro-lens array CSU 10 scan head. A Nipkow spinning disk confocal microscope that uses a single CCD camera to capture the image. The Yokogawa Nipkow disk-scanning unit (see page 435) is the "heart" ofthe PerkinEImer confocal microscope. This compact and carefully engineercd scan head is attached directly to thc microseope. The image can be viewed in real time through the eyepiece on the top of the Yokogawa scan head, or the image can be captured electronically using a CCD camera attached to the scan head. u Micro-Iens array Nipkow disk: The Y okogawa scan head contains two finely engineered spinning disks, the micro-lens array disk and the Nipkow (pinhole) disk. The micro-lens array disk contains a large number of very small lenses that focus the laser irradiating light into a corresponding "pinhole" that is spun in tandem with the micro-lens array disko Fixed "pinhole" Nipkow disk: The Nipkow disk contains a large number of pinholes that reject out• of-focus light. Due to the design ofthis instrument the size ofthe pinholes cannot be adjusted. Dichroic mirrors: Mounted within the Yokogawa scan head is a dichroic mirror (between the micro• lens array disk and the pinhole disk) for directing the fluorescent light towards the CCD camera. CCD c..ni The sensitivity and speed of the CCD camera attached to the Y okogawa scan head deterrnines the number of frames per second that can be collected in low light fluorescence imaging applications. The faster Ultra VIEW LC I FES 250 uses a CCD with frame transfer digital technology, resulting in high speed imaging of over 50 frames per second. Other cameras used by PcrkinElmer include interline CCD cameras for high quality, but somewhat lower speed image acquisition (up to 20 frames per second). These cameras provide 12-bit digitisation to create a wide dynamic range that is particularly useful for fluorescence imaging. The PerkinEImer assembled confocal microscope using the Yokogawa CSUIO scan head utilises a single CCD camera - with multiple imaging being perforrned by sequential scanning. '--s Lasers: Argon-ion Krypton-argon-ion, argon-ion / helium-neon combinations or other lasers are "-----"-= mounted separately from the scan head with the laser light being transferred using a fibreoptic connection. Laser power: The laser power can be adjusted by rotating Ihe koob on the laser casing. This controls the laser output - not necessarily the amount oflaser light reaching the sampie. AOTF laser attenuation: The amount of laser light is controlled by using software controlled AOTF filters (giving accurate levels ofbetween 0 and 100% ofthe current laser power setting). The Y okogawa scan head can be attached to most upright or inverted light microscopes. High-speed single channel fluorescence. Dual channel possible by attaching a second CCD camera. Simultaneous transmission imaging also possible if a bright-field CCD camera is attached to the microscope directly. Direct viewing ofthe confocal image is possible. Zoom, ROI, panning etc are not possible due to the way in which the image is collected via a CCD camera. Ti Transmission images can be overlaid with fluorcscence images if a CCD camera is attached to the microscope directly. The Yokogawa scan head controls are operated manually. The microscope is operated via a standard PC compatible computer. A single large computer screen is used to display the images as they are collected and the controls for scanning and changing microscope settings. The "basic" Ultra VIEW confocal microscope requires manual operation of thc Yokogawa scan head controls. Proprietary Perkin-Elmer software for controlling the microscope and CCD camera. The software can also perform basic image manipulation. Temporal Module: This software is for high-speed time• lapse imaging (including 4-0 and multi-labelling). Spatial Module: Software for high quality imaging of sampies with minimal movement or fluorescence changes. Ultra VIEW LCI is a fuHy computer controHed instrument with a single CCD camera. VItra VIEW is a basic system with manual filter changes. A variety ofconfigurations ofthe basic instrument are available, including a range ofCCD cameras. Appendix 1: Confocal Microseopes VisiTech 441 VisiTech International isiTech International supplies and manufactures a range of imaging related instruments for use in the life Vsciences. VisiTech manufactures two high-speed confocal microseopes that are particularly suited to live cell imaging. One instrument is based on the spinning Nipkow disk scan head manufactured by Y okogawa, and the other is a high-speed spot scanning system using an acoustic-optical deflector (AOD) to scan the laser across the sampie. VisiTech has a number of distributors, some of which make substantial contributions to the design and layout oftheir confocal microscopes, particularly the instruments based on the Yokogawa scan head. VISITECH CONFOCAL MICROSCOPES The VT-Eye confocal microscope (Figure A-54) is based on a scan head manufactured by VisiTcch International that uses a high-speed acousto-optical deflector (AOD) to scan a focussed laser beam across the sampie at very high speed. This instrument is particularly suited to live cell imaging where high-speed image acquisition rates and the imaging advantages of a confocal microscope are required. (- Figure A-54. VisiTech International VT -Eye Confocal Microscope. The VisiTech International VT-Eye confocal microscope is a high-speed laser scanning confocal microscope ideally suited to live cell imaging. Very high-speed seanning is achieved by using an aeousto-optieal deflector (AOD) device for scanning the laser in the x-direction. A conventional galvometric mirror is used for the y-scan direetion. The VT -Eye sean head is shown above attached to the side port of a Zeiss inverted research grade light mieroscope. This photograph is reproduced with perrnission from VisiTcch International, UK. VisiTech International Ltd. Web: www.visitech.co.uk/ Md",,, Unit 92 Silverbriar, Sunderland Enterprise Park (East) Sunderland. SR5 2TQ. UK Phone, +44 (0) 191 5166255 I FAX'I +44(0) 191 5166258 Appendix I: Confocal Microseopes 442 VisiTech VisiTech International Confocal Microscopes VisiTech International produces two high-speed laser scanning confocal microscopes. The VoxCell Scan confocal microscope, assembled using the spinning Nipkow disk scan head manufactured by Yokogawa, and the VT-Eye, which utilises a very high-speed acousto-optical detlector (AOD) to create a spot scanning confocal microscope capable ofvery high-speed scanning. VisiTe eh Instruments VT-Eye An acousto-optical detlector (AOD) based scanning mechanism resulting in a confocal (Figure A-54) microscope with unusually high scan speeds (30 frames per second for a 1024 x 1024 image), even though a single laser spot is used to scan the sampie. Up to 4 detection channels for multi-Iabelling applications. AOTF filters for laser line selection and operated using software running under Windows XP. The VT-Eye confocal microscope is based on the same technology as used in the Noran OZ confocal microscope, but with significant modifications made by VisiTech to the original design. VoxCell Scan Visitech International, and a number of distributors (listed below) put together a (Figure A-55) complete confocal microscope based on the Yokogawa CSUIO scan head (see page 435). The instrument, being based on the same CSUlO scan head as the PerkinElmer Ultra VIEW LC I confocal microscope, has many of the same specifications as the instrument assembled by PerkinEImer (see page 440 for details). However, due to the modular design of this instrument there are often important differences, not just between the VoxCell Scan and the Ultra VIEW, but also between each VoxCell Scan assembled. Various lasers are mounted on aseparate platform, with the attenuated laser lines of choice being transferred to the scan head by fibreoptic connection. Motorised optical filter wheels are often inserted between the scan head and the CCD camera. A dual camera attachment for the instrument is also manufactured by VisiTech to allow for simultaneous duallabelling. Distributors: VisiTech International has established a world wide distribution network by utilising a range of relatively small companies that have considerable technical expertise of their own. VisiTech, and a number of the distributors, often assemble a confocal microscope with specialised components as requested by the customer. This may inc1ude specialised CCD cameras, AOTF filters for laser line selection, optical filter wheels for multi-Iabelling applications and a nanofocus device for highly accurate positioning of the microscope objective. The following distributors use licensed components from VisiTech International, as weil as items sourced from other specialist manufacturers. Chromaphor Analysen-Technik GmbH (Germany) www.Chromaphor.de McBain Instruments (California, USA) www.mcbaininstruments.com Quorum Technologies Inc. (Canada) www.quorumtechnologies.com Solarnere Technology Group (USA) [email protected] Visitron Systems GmbH (Austria/Switzerland) www.visitron.de Appendix 1: Confocal Microscopes VisiTech 443 The VoxCell Scan confocal microscope (Figure A-55, with a short technical description on the previous page) is a spinning Nipkow disk instrument that utilises the QLCIOO Nipkow disk sc an head, which is based on the Yokogawa CSUIO scan head (see page 435). The VoxCell Scan is capable ofhigh-speed imaging due to the multi• pinhole design ofthe spinning Nipkow disk (see page 91). High-speed image acquisition is particularly suited to the imaging of dynamic processes in living cells. The micro-Iens array disk incorporated into the design of the Yokogawa scan head does mean that more light is focussed onto the sampie than when using the Nipkow disk alone (increasing the sensitivity of the instrument). However, compared to a conventional spot scanning confocal microscope, the amount of light per spot in a Nipkow disk based instrument is relatively low. The low excitation light level at the sampie results in significantly less photo-damage, both to the fluorophore (manifested as less photo fading) and to the integrity of the cells (allowing one to image live cells or embryos over many hours). The principles involved in the Nipkow spinning disk technology are described in more detail on pages 91 to 93 in Chapter 3 "Confocal Microscopy Hardware" and in the sections on the Y okogawa scan head (page 435) and the Ultra VIEW confocal microscope produced by PerkinEImer (page 438) in this appendix. Inverted light mlcroscope / Vlewlng port CCD cameras I \ /Y r Dual camera adapter Yokogawa scan head Figure A-55. VisiTech VoxCell Scan Confocall\1icroscope. The VisiTech VoxCell Scan confocal rnicroscope is a high.speed spinning Nipkow disk cOllfocal rnicroscope buih using the QLCIOO Nipkow disk ! micro-Iens array scan head manufactured by Yokogawa (see page 435). VisiTech also manufactures a dual camera adaptor (shown in the above photograph) for simultaneous dual channel imaging. This photograph is reproduced with perrnission from VisiTech InternationaL UK. Appendix I: Confocal Microscopes Glossary 8-bit: each pixel in an 8-bit (I byte) digital image conventional galvometric mirror is normally used to contains 256 (28) grey levels, usually from zero scan more slowly in the y-direction. (black) to 255 (white). Some image processing Airy Disk: diffraction pattern created by an object programs consider zero = white and 256 = black (Le. under the microscope. Rings of brightldark pattern the image will appear to be grey-scale inverted). All may be seen around small objects at high confocal microscopes can collect 8-bit images, and magnification. The Rayleigh Criterion is one way to many can also collect 12-bi images ifrequired. describe the limit of resolution of the microscope. This 12-bit: each pixel in a 12-bit digital image contains criterion describes the amount of overlap of 4096 (i 2) grey levels. Most confocal microscopes neighbouring Airy disks - when the two Airy disks have the option of collecting images in 12-bit. merge the object can no longer be resolved. 16-bit: each pixel in a 16-bit digital image contains Aliasing: the formation of artefactual objects or edges 65,536 (i6) grey levels. within a digital image that did not exist within the 3D stack: aseries of images collected on a confocal original sampie, a result of digital sampling errors. microscope by moving the focal plane in discrete z• Aliasing is avoided by sufficient sampling (see also steps with either a fine focus motor drive or a Nyquist criteria). piezoelectric z-stepper that moves either the A1ignment: the laser must be aligned properly into the microscope stage or the objective. objective, and the returning fluorescent light must also AID converter (ADC): an electrical device used to be aligned into the pinhole(s). In some confocal covert analogue signals to digital signals. microscopes these alignments are factory set and then Aberration: an optical error caused by an imperfect tested when the machine is installed. Other confocal optical system or specimen. See also "Chromatic microscopes require the user to periodically check the aberration" and "Spherical aberration". alignrnent. If the sensitivity of the machine appears Absorption curve / spectrum: the absorption of tower than expected then check the alignment specific wavelengths (colours) of light, normally (particularly the alignrnent of the laser into the plotted as intensity verses wavelength. Each confocal pinhole). fluorophore has a characteristic absorption profile. In AM: see "Acetoxymethyl ester". single photon excitation the absorption spectrum is the Analogue: a continuously variable electrical signal. same as the excitation spectrum for a given Analogue-to-Digital converter: see "AlD converter". fluorophore. Analyser: a light polariser filter used in DIC Acetoxymethyl ester (AM): a chemical derivative of (Differential Interference Contrast) microscopy. many common fluorescent dyes which makes the Angstrom (A): a unit of length, 1/l0th of a nanometre molecule non-charged, thus allowing the fluorophore (10. 10 meters). to more readily diffuse across the cell membrane. The Annulus: see "Phase Annulus". methyl ester is removed by esterases naturally present Antifade: compounds such as n-propyl-gallate and p• inside a living cell, thus trapping the dye within the phenylenediamine help slow down the rate of fading cell. An added bonus for fluorescent labelling is that (or photobleaching) of the sampie. Antifade reagents the AM-ester derivative of the fluorophore is often are antioxidants, as photobleaching is due to light non-fluorescent, only becoming fluorescent on induced oxidising reactions. Lowering the amount of c\eavage of the AM ester group within the cell. laser light reaching the sampie or limiting the amount Achromat: (also achromatic) a lens designed to reduce of oxygen available also helps to reduce fading. chromatic aberration of the red and blue wavelengths Antioxidant: molecules that tower the level of and spherical aberration of green wavelengths. oxidative damage to the cell or tissue by reacting with Acoustic-Optical Tuneable Filter: see "AOTF". free radicals that are created by the illuminating light. Acousto-Optical Deflector (AOD): high-speed laser Antioxidants will lower the amount of fading of the scanning can be achieved in a confocal microscope by fluorophore. using an AOTF type active crystal for scanning the Anti-reflection coating: a special coating on air-glass laser beam at very high-speed in the x-direction. A surfaces of optical components (lens, optical filters Con(ocal Microscopy for Biologists. Alan R. Hibbs. Kluwer Academic I Plenum Publishers. New York, 2004. 444 Glossary 445 etc) that minimises the amount of light reflected at the (i.e. high NA objectives have correspondingly higher surface interface. axial resolution). AOTF (Acoustic-Optical Tuneable Filter): is an Back Cocal plane (BFP): the focal plane of a lens active crystal device that acts as a variable diffraction located on the side of the lens away from the object. grating. Varying the radio-frequency acoustical Phase rings are inserted at the BFP. The BFP can be vibrations in the crystal allows one to diffract out a viewed using a Bertrand lens or phase telescope. desired wavelength of light. An AOTF filter can be Backscatter: a term used to describe the light that is used to select one or multiple wavelengths from a scattered by the specimen back towards the objective multi-Iine laser, and to adjust the power level lens. The confocal microscope can create an excellent transmitted (a continuously variable "neutral density" high-resolution image using "backscattered" light filter). (sometimes referred to as "reflectance" imaging). Aperture diaphragm: an adjustable diaphragm located Band Pass filter (BP): an optical filter that transmits a in the condenser optics of the microscope. Used to band of colour, the centre of which is the centre adjust the effective NA ofthe condenser. wavelength (CWL). The width ofthe band is indicated Apochromat: (also Apochromatic) a lens in which by the full width at half maximum transmission chromatic aberration has been corrected for three or (FWHM), also known as the halfbandwidth (HBW). more colours (red, green, blue and UV). Also Barrier filter: see "Emission filter". corrected for spherical aberration for two or more Beam splitter: a partial mirror, polarisation filter, prism wavelengths (green and red). or diffraction grating that diverts part or all of the light Astigmatism: an optical aberration that results in the from one direction to another. A beam splitter is often horizontal and vertical focus not coinciding along the used to divert some or all of the light from the optical axis (resulting in a circular object appearing eyepieces to the camera, to select specific wavelengths ellipsoid). for irradiating the sampie in a confocal microscope, or Autofluorescence: the inherent ability of a specimen to for directing specific wavelengths of light to fluoresce. In mammalian cells the principle cause of individual channels (PMTs) in a confocal microscope. autofluorescence are flavin coenzymes (FAD, FMN The primary beam splitter in a confocal microscope and NADH). In plant cells lignin (green fluorescence) splits the irradiating laser light from the returning and chlorophyll (red fluorescence) are the major fluorescent light. sources of autofluorescence. Aldehyde fixatives Bertrand lens: a lens that allows the back focal plane induce fluorescence in biological material, which is ofthe objective to be viewed through the eyepieces. often incorrectly called autofluorescence. Binary image: an image comprised of only 2 intensity Autumn LUT (Bio-Rad): red colour "Look Up Table" values - usually represented by black and white. used by Bio-Rad (dark red - light red - yellow - white Bit: the basic unit of digital information ("binary unit"), colour gradient) for colouring digital images. conveys 2 possible states denoted on/off or 1/0. Averaging: multiple images are averaged to reduce Bit depth: see "Dynamic range". Poisson noise, such that each pixel intensity in the Black level: the amount of signaloffset (alters the final image is an average of intensities from the blackness level ofthe image). corresponding pixels in the constituent images. Bleaching: see "Photobleaching". A vidin: a 66KDa glycoprotein derived from egg white Bleed-through (bleed-over): occurs when the emission that specifically binds biotin. Biotinylated probes are of one fluorophore (e.g. ethidium bromide) is also detected with fluorescently labelIed avidin. The detected in the second channel (e.g. the fluorescein bacterial equivalent is called Streptavidin. channei). There is always some bleed-through Axial illumination: occurs in conventional bright-field (minimised by careful setting of the gain control), but imaging when the condenser aperture is closed down. unwanted bleed-through can be lowered considerably Increases the contrast and depth of field. Not by suitable choice of optical filter blocks. The Bio• recommended for high resolution, but is useful for Rad software allows on screen "mixing" of each of the "finding" the specimen. detection channels, thus allowing one to subtract a Axial resolution: resolution along the z-axis, Le. predetermined percentage of one channel from the resolution perpendicular to the plane of focus. In other to minimise the effects ofbleed-through. confocal fluorescence microscopy the axial resolution BMP: Windows Bit Map, a standard image format for increases inversely proportional to the square of the Windows-compatible computers. You can also specify NA of the objective - in contrast to lateral resolution, RLE compression. which is to the first power of the NA of the objective 446 Glossary Box size: size of the image collection box in pixels. avoids the problem of chromatic aberration, but multi• Commonly used sizes for LSCM are 512 x 512, 768 x channel imaging on a confocal microscopy may result 512 or 1024 x 1024 pixels. in misregistration between images made with Bright-field: this is the "normal" wide-field fluorescent dyes that emit at different wavelengths. illumination method in microscopy when viewing a CLSM: see "Confocal Laser Scanning Microscope". specimen down through the eyepieces (see Köhler C-mount: standard screw-in lens mount for the illumination). In the absence of a specimen, the attachment of a camera to a microscope (or other background appears "bright". See also, "Dark field" device). and "Epi-illumination". Coherent light: light consisting of waves vibrating in Byte: a unit of information storage employed in a the same phase, but not necessarily the same plane. computer, comprised of 8-bits. Laser light is monochromatic, linearly polarised and Caged probes: molecular probes that are converted to highly coherent. their active derivative upon irradiation with UV light. Collector lens: a lens used to collect light from an In this way highly reactive species can be targeted to emitting light source and to pass the light through the subcellular destinations before being activated. field diaphragm and the aperture diaphragm to the CCD and CID: electronic chips as the detector in an specimen. electronic camera. CCD = charge-coupled device, CID Colour Look Up Table: see "Look Up Tables". = charge-induced device. Both cameras use an array of Colour saturation: colour saturation refers to the photodiodes on a chip which record an image as a degree of colour present (i.e. intensity of colour). mosaic of charges. Colour temperature: a measure of the bluish (high CD "burner": a device for writing information to CD• temperature ) or reddish (Iow temperature ) hue of the R disks, also known as "CD-ROM disks". The "white" light expressed as the absolute temperature "bumed" disk can then be read in a regular CD-ROM (degrees Kelvin). This nomenclature is used to express drive. A disk can be written as a single session or both the sensitivity of an electronic light detector and several times as a multi-session disko the colour display of a computer monitor. CD-R: Recordable CD (optically written disk) of 650 or Compound microscope: produces magnification using 700 MB capacity. Also known as CD-ROM (Compact an objective and an eyepiece - resulting in an upright Disk - Read Only Memory). Once data has been image. written to the disk, the data can be read and copied, Compression: see "File compression". but cannot be erased or modified. However, re-writing Condenser: lens system usually under the stage, but in to a multi-session disk may result in previous files of an inverted microscope the condenser resides above the same name no longer being accessible (the the stage. This lens is designed to focus and project directory information is changed). illuminating light onto the specimen. In epi• CD-RW: Re-writable CD laser disk, with the same illumination, as used in both wide-field epi• capacity as the CD-ROM (650 or 700 MB), but can be fluorescence microscopy and laser scanning confocal writtenlerased many times. Although the CD-RW disk microscopy, the objective itself acts as both condenser can be read using the CD-ROM drive present in most and objective. With the confocal microscope a computers, reading the disk on other computers from transmission image can be obtained by collecting the which it was originally written may require the laser light that passes through the condenser. installation of additional software. Condenser aperture: a variable aperture (iris) in the Centre wavelength (CWL): the arithmetic centre ofthe condenser used in Köhler illumination to vary the window of transmission of a Band Pass filter. This numerical aperture ofthe illumination. value is not necessarily the same as the peak Confocal: an image of only the in-focus plane, obtained wavelength. by passing the retuming light through a pinhole. Channels: different wavelengths (colours) of light are Confocal aperture (pinhole or iris): defining feature collected in separate "channels" in a confocal of a confocal microscope. To reach the detector, the microscope. Each channel will have its own set of light retuming from the sampie is focussed through an discriminating filters and light detector. aperture that removes any out-of-focus light rays, Charged-Coupled-Device: see "CCD". thereby producing an "optical section". In Bio-Rad Chromatic aberration: glass refracts different colours instruments the confocal iris is millimetres in size, (wavelengths) of light to different extents. Most compared to a very fine micron sized iris for most microscope lenses use combinations of elements to other confocal microscopes. The Nipkow disk based minimise chromatic aberration. Monochromatic light confocal microscopes have a large array of pinholes Glossary 447 within the disk that allow only in-focus light to Cryo-fixation: rapid cooling of a specimen to preserve penetrate the disko its structure (usually to liquid nitrogen temperatures). Confocal Laser Scanning Microscope (CLSM): uses Cryostat: a trade name for a refrigerated rnicrotome a scanning system consisting of rotating mirrors or used for cutting frozen sections. opto-acoustic deflectors to scan a point of laser light Curvature of field: when the focal plane of a lens is over the specimen. curved instead of flat. Conjugate planes: planes in a complex optical system Cyan: a light blue colour forrned when red light is at which a reference plane is mutually in focus. subtracted from white light (leaving green + blue). Constructive interference: phenomenon of increased CMYK (Cyan, Magenta, Yellow and Black) inks are light intensity that results when light waves are in used in most printers. step, travelling and vibrating in the same direction and Dark current: the background current produced by a are at the same location at the same time. photodetector. Cooling a CCD or PMT detector Contrast: the degree of visibility between two objects significantly reduces the dark current noise. or of an object against its background. A low-contrast Dark field illumination: illumination that passes feature blends into the background, and a high• through the specimen at such an angle that light rays contrast feature stands out distinctly. Contrast can be cannot enter the objective lens directly. Dark field manipulated during acquisition by altering the gain images have a black background on which structural and blackness level on the instrument, as weil as features appear lighter. Useful for observing live changed after collection by subsequent image unstained cells. processing. Deconvolution: mathematical process used to improve Contrast range: see "Dynamic range". the elarity of images by re-allocating out-of-focus Contrast stretch: a process applied to digital images light back to its plane of origin. Deconvolution can be that remaps intensities in the collected image to spread perforrned on a z-stack obtained from a wide-field them over the maximum possible range of values fluorescence, confocal or multi-photon microseope. (increases contrast within the image). Depth of field: the thickness of the optical slice (the Cooled CCD: a CCD camera that operates below distance along the z-axis of the specimen that is in• ambient temperature to reduce or eliminate dark focus). High numerical aperture (NA) objectives have current "noise". a very small depth of field resulting in a very thin Cooling fan: argon-ion and krypton-argon-ion lasers optical slice when using a confocal microscope. Not to are air cooled by a fan. The fan must be left on for be confused with "depth of focus", which is the z• several minutes after tuming the laser off to ensure the distance in the image plane of the microscope that is laser has cooled sufficiently. in focus. Correction collar: an adjustable ring on an objective. Depth of focus: the thickness of the image plane that is There are two types, one controlling a diaphragm in in focus. In a high NA lens the depth of focus is large the back focal plane which allows the NA to be (the position of the eyepiece is not critical), but the adjusted when setting darkfield illumination, and the "depth of field" (the physical z-distance within the other allowing compensation for different immersion specimen in focus) is very small. media or coverslip thickness (this collar can also be Destructive interference: is where light waves are out used to correct for spherical aberration created by the of step - resulting in decreased light intensity. If the sampie). two waves are out of step by 1/2 wavelength then the Coverslip thickness: the correct coverslip thickness peak of one will exactly co-inside with the trough of (usually 0.17 11m, number 1.5 coverslip) is important the second and result in zero intensity if the two initial for optimal resolution, and in fluorescence confocal light rays have the same intensity. microscopy also for optimal sensitivity. Coverslip DF (Discriminating Filter): an optical filter with very thickness is especially critical when using a "dry" lens. steep-sided transmission windows, with especially The required coverslip thickness is engraved on the deep attenuation of energy elose to the band. For side ofthe objective. example, 488DFlO, describes a Discriminating Filter Critical illumination: a type of illumination in which (DF) that allows a narrow band of 10 nm light, in the the image of the light source is focussed onto the vicinity of 488 nm, to pass through. specimen. This type of illumination requires light Diascopic illumination: light transmitted through the scramblers to avoid having a pattern of the specimen, using a condenser to focus the light. illumination source on the image plane. DIC microscopy: see "Differential Interference Contrast". 448 Glossary DIC prism: see "Wollaston prism". Dry objective: an objective that is used without Dichroic mirror: (also dichroic beamsplitter) a mirror immersion media (i.e. only an air gap) between the that reflects certain wavelengths and allows others lens and the specimen. Highly susceptible to spherical through unhindered. They can be made with one, two aberration, and must be used with the correct coverslip or three reflective ranges (singe, double and tripIe thickness (some objectives have an adjustment collar dichroic mirrors). For example, the tripIe dichroic for differences in the thickness ofthe coverslip). mirror in the Tl filter block of the Bio-Rad MRC- Dual imaging: the ability to collect two images from 1024 reflects a region of light around 488, 568 and two separate light detecting channe1s on a confocal 647 nrn - light not reflected passes through this microscope. mirror. Duallabelling: the labelling of a sampie with two Differential Interference Contrast (DIC): (Nomarski differently coloured fluorophores so that the co• optics) is an illumination technique in microscopy localisation of the two structures/molecules of interest where polarised light is used to produce an apparent can be deterrnined. 3D effect by creating light and dark shadows at DVD disk: very high capacity laser disk (4-15 x the opposing edges of features in the specimen. Excellent capacity of a CD-R disk). Re-writable DVD disks are DIC images can be collected using the transmission available, but there are currently several formats and channeIon a confocal microscope equipped with DIC standards for DVD disks. optics. The "shadows" in the DIC image are created by DweU time: the amount of time the excitation light small changes in refractive index, and are not a true illuminates a spot in the sampie, and hence the 3D representation ofthe sampIe. collection time for each pixel in the sampie. A longer Diffraction: a change in the direction of light caused by dweil time may result in more photo damage to the the interaction of the light with an object. Diffraction sampie. Typical dweil times for a laser scanning is greatly increased when light passes objects that are confocal microscope are 0.1 to 1.0 !-Is. elose to or smaller than the wavelength of light. Dye saturation: refers to the highest level of light that Diffraction grating: aseries of parallel lines, forrning can be used to produce fluorescence - further increase alternating grooves and ridges with spacings elose to in the laser light intensity will not increase the level of the wavelength of light, scribed on a reflecting or fluorescence. transparent substrate. A mixture of various Dynamic range (or Bit Depth): the number ofpossible wavelengths, including white light, can be readily split shades of grey between black and white in an image. into the component colours using a diffraction grating. An 8-bit image has 256 (28) possible grey levels, a 12- The Meta channel on the Zeiss 510 LSM confocal bit image has 4096 (i2) fossible grey levels and a 16- microscope uses a diffraction grating to separate the bit image has 65,536 (2 1 ) possible grey levels. fluorescent light into its component colours. Edge enhancement: an algorithm for sharpening the Digital: the collection and/or storage of information as a edges on features within digital images. series of numbers. Images from confocal microscopes Edge Filter: another term for a short pass or long pass are stored as digital image files. optical filter with a very sharp cut-on or cut-off. Digital zoom: zooming (enlarging) the image by EFLP (Long Pass Edge Filter): an optical filter that spreading the available pixels over a larger area - this reflects more than 99.999% of shorter wavelength may result in a pixilated image showing relatively light, with a very sharp cut-offwavelength. large pixels, or a srnooth image in which the enlarged ELF (Enzyme Labelled F1uorescence): substrates that pixels have been "smoothed" using a computer yield fluorescent precipitates at the site of enzymatic algorithm. Digital zoom does not result in increased activity. resolution. Also see "Scanning zoom". Emission: release of light from a fluorophore when an Diopter setting: the facility for focusing one eyepiece excited electron returns to the ground state. separately from the other to compensate for Emission fIlter: an optical filter that ensures that only differences in focus between the microscopist's eyes. light of the desired wavelengths reaches the eyepieces Dispersion: the property of light whereby it splits into or CCD camera of a wide-field epi-fluorescence separate component colours when undergoing microscope, or the light detectors of a confocal diffraction, refraction etc. (i.e. the amount of microscope. diffraction, refraction etc. varies with wavelength). Emission fingerprinting: the process of collecting DRLP: "Dichroic Long Pass" optical filters that spectral information from the sampIe and applying transmit a broad range of longer wavelengths of light, linear unmixing to separate fluorophores with highly while efficiently reflecting shorter wavelengths. overlapping emission spectra. Glossary 449 Emission spectrum: the spread of wavelengths over light path (usually quoted at the wavelength of which the fluorophore emits fluorescent light. The maximum absorption). radiation results from electrons returning from the first Eyepiece graticule: a transparent disk marked with a singlet excited states to ground level. The fluorescence scale or other measuring device that fits into the emission wavelengths in single photon excitation are eyepiece at the level of the intermediate image, and is always of longer wavelength (towards the red) therefore superirnposed on the image of the focus compared to the wavelength used for excitation. plane. Used for approximate measurements in Emission wavelength: usually the wavelength of microscopy. Different scales are needed for different maximum emission (519 nm green light for magnification objectives. fluorescein). See also "Peak emission wavelength". Eyepieces (oculars): are lenses that project the Empty magnification: greater magnification than is intermediate image through the optics of the eye to useful. No increase in resolution, and sharpness and form an image on the retina. A typical eyepiece has a contrast will decrease. This concept once depended on magnification of 10x, but lower (5x) and higher (20x) the acuity of the eye, but now depends on the size of oculars are common. The oculars are not used when the image pixels relative to the resolution limit of the scanning the laser in a confocal microscope to microscope/lens being used (>2.5 pixels/resel). generate an image. Epi-illumination (epi-fluorescence): excitation light is FACS (FIuorescence Activated CeU Sorting): is a reflected off a 45 degree dichroic bearnsplitter and machine that sorts (or counts) individual fluorescently through the objective to the specimen. Fluorescence or tagged cells (normally using fluorescently conjugated reflected light is detected after passing back through cell specific antibodies) using a laser. Similar the objective lens, and directly through the dichroic fluorochromes to those used in confocal microscopy beam splitter. The objective therefore also acts as a (such as FITC, TRITC etc) can be used in a FACS condenser. Conventional fluorescence microscopy, instrument. confocal fluorescence microscopy and back scatter Fading: see "Photobleaching". imaging are all performed using epi-illumination. False colour: all colour displayed in digital images Esterase derivatives: see "Acetoxymethyl (AM) ester". collected on a confocal microscope is "false" colour - Excitation: absorption of energy in the form of light Le. the colour is added by the computer, and does not causes an electron to jump from the ground state to a directly reflect the range of wavelengths of light higher energy level (the excited state). The subsequent directed to that particular channel. drop back to the ground state results in the emission of Fast X-V: fast scan speed on the Zeiss LSM 510 for a photon of longer wavelength (lower energy) than locating the sampie (increased y-scan speed, and that of the original photon that excited the electron. decreased lines, resulting in larger pixels). Excitation mter: an optical filter (usually of the Fibreoptic coupling: the use of fibreoptic cable to interference type) which limits the incident light used connect various lasers to the confocal microscope scan to excite the fluorochrome to specific wavelengths. head, and on some instruments to direct the Excitation spectrum: the spread of wavelengths of fluorescent light from the scan head to an optics box light that can be used to excite the fluorophore. where the light is separated into individual channels. Excitation wavelength: the wavelength of peak Field aperture: see "Field diaphragm" . excitation for a fluorophore (for example, elose to 488 Field curvature: an optical distortion where the centre nm for fluorescein). of the image is in focus and the edges remain out of Exit pupiI: the point just above an eyepiece where an focus. A high quality "plan" objective will have a image is formed that can be viewed by placing the "flat" field of view, but at a penalty of more elements surface of the cornea of the eye at that position. and less throughput (transparency). Out of focus areas Extended focus image: aseries of optical slices can be of an image will not be "seen" by confocal microscopy collected and displayed as a single composite image - resulting in an image truncated around the edges (simply added together or projected in a variety of when field curvature is present. A lens with field different ways - see "Projection"). An extended focus curvature can often be used successfully in confocal image is usually only useful for sampies with a microscopy by using the centre of the field of view relatively small number of fluorescent structures. only (Le. zooming the image). Extinction coemcient (e): The amount of light Field diaphragm: an iris controlling the size of the absorbed by a particular molecule. The molar illuminated field in the sampie. Usually located elose extinction coefficient is the optical density of a one to the light port (between the condenser and the light molar solution of the compound through a one cm 450 Glossary bulb). In Köhler illumination the field diaphragm life of 10-8 seconds) only as long as the irradiating should be focussed at the same plane as the specimen. light is present. Field iris: see ''Field diaphragrn" . Fluorescence microscopy: a form of light microscopy File compression: used to reduce the storage space using fluorescent dyes to highlight the area of the required by an image data file. Lossless techniqucs specimen of interest. Conventional (wide-field) epi• compress image data without removing detail (but fluorescence microscopy is carried out using a may lose colour information); lossy techniques mercury or xenon arc lamp. Laser scanning confocal compress images by removing detail. microscopy usually, but not always, uses fluorescence Filter (optical): an optical device that may change the imaging to detect the region of interest. intensity of light, or block specific ranges of Fluorescence Recovery After Photobleaching: see wavelengths of light from passing through the filter. "FRAP". Optical filters may be simple coloured glasses, Fluorescence Resonance Energy Transfer: see interference filters or tuneable acoustic-optical "FRET". devices. Fluorescent probe: a fluorochrome, i.e. a molecule that DF= Discriminating Filter LP= Long Pass exhibits fluorescence. SP= Short Pass KP= Kurz (short)Pass Fluorite: (CaF2) a type of "glass" used in some DRLP= Dichroic Long Pass EFLP= Long Pass Edge Filter NB= Narrow Band OG= Orange Glass microscope objectives, particularly for use in UV RG= Red Glass WB= Wide Band fluorescence microscopy. Fluorite lenses are only ND= Neutral Density partially colour corrected. Not suitable for polarisation AOTF= Acoustic-Optical Tuneable Filter microscopy. AOBS= Acoustic-Optical Band Selection AOD= Acoustic-Optical Deflector Fluorochrome: a molecule that exhibits fluorescence. FISH (Fluorescence In Situ Hybridisation): a Also known as a "fluorescent probe". technique used to locate genes on specific Fluorophore: the part of the molecule responsible for chromosomes, and to map genes relative to known fluorescence. Fluorophores, such as fluorescein, genetic markers. rhodarnine etc are often attached to other molecules Fixation: a process of cross-linking (mainly aldehyde such as antibodies to create a fluorescent probe, of fixatives) or precipitating (organic solvents) the which the fluorescein or rhodamine moity is the protein constituents of the cell to retain the structural fluorophore, and the whole molecule is known as a integrity of cells. Care should be taken to retain not fluorochrome. only 2D structural integrity, but also the 3D volume of Focallength: the distance between the optical centre of the cell if 3D confocal microscopy is to be attempted. the lens and its focal point. Each lens has a focal point Glutaraldehyde, although resulting in excellent on each side ofthe lens. structural integrity, does create a high level of Focal plane: an imaginary 2-dimensional plane at right autofluorescence. angles to the optical axis and passing through the focal FLIM (Fluorescence Lifetime Imaging): is a powerful point. The focal plane can be thought of as the technique for the analysis of molecular interactions imaginary "screen" on which the image is formed. See using FRET. The lifetime of the fluorescence is also "back focal plane" and "front focal plane". measured directly, which means FRET interactions Focal point: the point on the optical axis at which light can be monitored independent of the concentration of entering the lens parallel to the optical axis comes to a the individual molecules under study. focus. FLIP (Fluorescence Loss in Photobleaching): is used Focus: the ability of a lens to converge light rays to a to study the molecular dynamics and connectivity of single point. A high quality colour corrected lens will cellular membranes. result in all wavelengths being focussed to a single Flow cytometry: see "F ACS, Fluorescence Activated point. The size of the "point", known as the "Point Cell Sorting". Spread Function", is determined by the NA of the lens Fluorescence: the emiSSIOn of light of specific and the wavelength of light (diffraction limited wavelengths from a molecule that is irradiated with microscopy) . light of a shorter wavelength (single photon Focus motor: computer controlled motor to move the excitation). Multi-photon excitation results in shorter fine focus (or sometimes the course focus) on the wavelength fluorescence emission after irradiation microscope, used for collecting aseries of optical with high-energy pulsed longer wavelength infrared slices. light. Fluorescence emission continues (with a half- Glossary 451 Fourier space: aspace containing the Fourier transfonn GlowUnder and GlowOver (Leica): Look Up Table infonnation of the object. The back focal plane of the algorithm used by Leica confocal microscope software objective is a Fourier space. to highlight areas of the image that are at the Fourier transform: the separation of an image into its maximum and minimum intensity level - used to spatial or frequency components. The Fourier accurately set the upper and lower light levels (gain transfonn image can be filtered and manipulated to and offset control). alter or reduce periodic signals within the image, and Glycerol immersion objective: an objective designed then an inverse transfonn function perfonned to regain for use with glycerol to fonn a continuum between the the original image (with selected frequencies lens and the coverslip. Often used for glycerol accentuated or removed). mounted specimens (particularly immunolabelling) to Frame grabber: (frame store) a computer card that reduce spherical aberration. Multi-variable (oil, allows images from video cameras, PMT tubes etc glycerol, water) immersion lenses are also available. (analogue outputs) to be digitised and stored as a Graticule: see "Eyepiece graticule". computer file. Green Fluorescent Protein (GFP): is a very powerful FRAP (Fluorescence Recovery After Photobleaching): als method of "tagging" cellular proteins with a naturally o known as Fluorescence Photobleaching Recovery fluorescent protein derived from the Jellyfish (FPR). This is a quantitative fluorescence optical Aequorea victoria. The DNA sequence coding for technique used to measure the dynamics of 2D GFP can be readily attached by gene fusion to any molecular mobility. protein of interest, and the fate of the protein followed FRET (Fluorescence Resonance Energy Transfer): is in real time in living cells. A number of derivatives of a technique for studying molecular associations that the original protein that have different wavelengths of are beyond the limit of light microscopy. The energy emission, have been developed. These include RFP absorbed by a fluorophore (donor) is transferred to an (Red Fluorescent Protein), YFP (Yellow Fluorescent absorber (acceptor) molecule which then emits light at Protein), CFP (Cyan Fluorescent Protein) etc. Also see a longer wavelength. The fluorophore (donor) and the "Reef Coral Fluorescent Protein" (RCFP). absorber (acceptor) molecules must be within a few Green LUT: green "Look Up Table" used to colour nanometres of each other, allowing one to study images with a gradient from dark green through molecular associations using light microscopy. yellow to white (often used to display green Front Focal Plane (FFP): is the focal plane of a lens fluorophores such as fluorescein). located on the side ofthe lens towards the object. Grey levels: number of actual intensity values present Gain: the level of signal amplification (alters the in an image. This is the product of the number of whiteness level of the image). Used in conjunction sampling events (photons absorbed), Poisson noise with the offset control (or black level) to produce an and electronic noise. The maximum number of grey• image with the best contrast. levels may be 256 (8-bit), 4096 (l2-bit), 65,536 (16- Gamma: used to describe the relationship between the bit) or even higher. original signal and the display image. A gamma of 1 Heat filter: an optical filter that blocks infrared indicates that the relationship is linear. Changing the radiation, but transmits visible light. They can be gamma can highlight areas of the image that show either absorption or interference (reflection) filters. particularly low fluorescence intensities. Histogram: see "Image histogram" . GEOG LUT: multi-coloured "Look Up Table" that has IFA (Immunofluorescence Antibody Assay): see series of bands of colour ranging from cool colours "Immunofluorescence" . (blue) to hot colours (yellow and white). Often used to Image: An array of intensity values representing light highlight areas of interest within the image. intensity at spatial or temporallocations in the sampIe. GFP: see "Green Fluorescent Protein" . Image analysis: perfonning measurements on features GIF (Graphics Interchange Format) is a file fonnat in a stored image without chan ging the image itself. commonly used to display indexed-colour graphics Image histogram: a bar graph depicting the number of and images in hypertext mark-up language (HTML) pixels at each grey level within the total image, or documents over the World Wide Web and other online within a selected region of the image. The image services. GIF is an LZW-compressed fonnat designed histogram can be used to both analyse the distribution to minimize file size and electronic transfer time. of fluorescence within the image and to manipulate Gigabyte (GB or Gbyte): a unit of storage capacity, the Look Up Table (LUT) levels within the image. 1,073,741,824 (230) bytes. 452 Glossary Image mathematics: image processing where whole Infrared (IR): light from the region of the spectrum images are added, subtracted, multiplied or divided with wavelengths between 750 nm (red) and 0.1 mm into one another. (rnicrowave). Image processing: altering the image to improve Intensity oflight: the flow of energy per unit area. contrast, sharpness, feature extraction etc. Intensity is a function of the number of photons per Image segmentation: the partitioning of a digital image unit area and their energy (the shorter the wavelength into non-overlapping regions according to grey levels, the higher the energy ofthe photon). texture etc. Interference: the interaction between one wave and Immersion media: the material used between the another, resulting in the addition or subtraction oflight objective and the sampie (air, water, glycerol, oil etc). in the overlapping area. Constructive interference is The type of immersion media used is marked on the when two waves are in step, travelling and vibrating in objective (no marking indicates air). Some objectives the same direction, at the same time in the same have a moveable collar that allows for adjustrnent for location - resulting in greater intensity. Destructive different immersion media. interference is where the waves are out of step - Immersion oll: an oil with a refractive index of 1.515 resulting in less intensity. If the two waves are out of that is used to provide a continuum of the same step by 1/2 wavelength then the peak of one will refractive index between the objective front lens and exactly co-inside with the trough of the second and the coverslip. Different immersion oils are not usually result in zero intensity. miscible - if you change immersion oil make sure the Interference fIlter: an optical filter that contains a lens is c1eaned well before applying the new oil. The series of optical coatings or layers that filter light by stated refractive index of the oil is at a specific causing reflectance or destructive interference of temperature (usually 23°C). specific wavelengths. Immunofluorescence (lFA): "Immunofluorescence Interpolation: the addition or subtraction of pixels in Antibody Assay" (also known as immunolabelling) is an image to reduce aliasing effects when changing the where antibodies are bound to cells or tissue slices and image size. Generally preserves shapes by altering are visualised by using fluorescently labelled intensities in replicated or remaining pixels. secondary antibodies, or by directly labelling the Inverted microscope: used for examining specimens in antibody with a fluorescent probe. Immunolabelling Petri dishes or incubation chambers. The condenser is provides a highly specific way of identifying the mounted above the stage with the objectives subcellular location of either the molecule of interest, underneath. Modern inverted rnicroscopes produce or of the organelle or subcellular structure where the images as good as those ofupright rnicroscopes. Using molecule is known to be located. a confocal scan head on an inverted microscope is Indium Tin Oxide (lTO): an electrically conductive, optically the same as using an upright rnicroscope. optically c1ear coating used on coverslips for heating Isobestie point: a wavelength point in an absorption or across the surface of the coverslip. ITO coated fluorescence emission spectrum at which the indicator coverslips are used in the Bioptechs heated chambers. fluorescence is insensitive to ion binding (at this point Infinity optics: most modern rnicroscopes now use the amount of absorbance or fluorescence is directly infinity corrected objective lenses (parallel light rays) related to the dye concentration and is not influenced rather than the older method of having a fixed focal by the binding of specific ions). length. The Bio-Rad confocal microscope scan-head Jaz disk: a relatively high-speed, reasonably high also uses infinity optics, allowing the "pinhole" to be capacity (1 to 2 Gbyte) removable magnetic disko located physically distant from the objective, and to be Convenient for daily use, but expensive for long term ofrelatively large, and variable, size (in millimetres). storage. Available for SCSI, parallel and USB ports. Infinity-corrected objectives: have a tube-Iength of JPG (JPEG): the Joint Photographic Experts Group infinity and so require aseparate lens to form an (JPEG) format is commonly used to display image. Their advantage is that the light from the photographs and other continuous-tone images in image is kept as parallel rays until just before the hypertext mark-up language (HTML) documents over eyepieces which means that optical elements can be the World Wide Web and other online services. introduced any-where along the light path without Unlike the GIF format, JPEG retains all colour affecting the focus or magnification of the final image. information in an RGB image but compresses file size Most modern microscopes now use infinity corrected by selectively discarding data. A JPEG image is objectives. automatically decompressed when opened. A higher level of compression results in lower image quality. Glossary 453 Kaiman averaging: an averaging algorithrn which movements (for example, when imaging live cells) displays a "running average" as images are collected. will not detract unduly from the quality ofthe image. Each image of the averaged set contributes a weight to Line scanning: continual scanning of a single, fixed• the final result that is proportional to its position in the line, can be used for rapidly monitoring the level of scan order. Kaiman averaging greatly improves the fluorescence. The image can be collected as a line• signal to noise ratio in the image, and can also be used time image in which the horizontal (x) axis represents to follow movement - where the latest images a physical line across the sampie, but the vertical (y) collected create a "ghosting" effect where objects have axis represents time - i.e. each subsequent line moved. collected is displayed one below the other. KG: a short pass colour absorption glass that transmits Line selection: some lasers have several specific visible light while attenuating both longer and shorter wavelengths of light available (Iines). A laser line wavelength energy. selection filter wheel, AOTF filter or AOBS filter Kilobyte (Kß or Kbytes): 1024 (2 10) bytes, 1000 Kb allows one to select individual lines or combinations equal to 1 Mb (megabyte). oflines for multi-Iabelling applications. Köhler illumination: the most common type of Linear unmixing: a mathematical process whereby illumination used in transmitted light microscopy, fluorescent light can be re-allocated back to the resulting in an evenly illuminated back focal plane of correct channel in multi-Iabelling applications. the objective for maximum resolution and an evenly Local area contrast: computer algorithrn that increases illuminated background. Developed by the German image contrast over a small area. Can be useful for biologist named Köhler (1866-1948). enhancing very small differences in intensity, but may Kompenzatione (K): notation located on the eyepieces dramatically increase any spotty or patchy areas of the of some microscopes, indicating that the eyepiece is image. responsible for some of the colour correction of the Local contrast enhancement: image-processing objective lens. German for "compensating". algorithrns that increases image contrast and sharpness Kurz pass (KP): (German for Short Pass) an optical over a selected area. filter that allows short wavelengths to pass. For Long Pass filter: see "LP" example KP 490 would indicate that all wavelengths Long Working Distance (LWD): an objective that has shorter than 490 (violet) pass through, whereas longer an unusually long distance between the objective lens wavelengths are blocked. front element and the object (coverslip). This type of Laser emission: all lasers emit light at discreet lens is especially useful for tissue culture and wavelengths (laser lines). Different lasers emit at microinjection work. A long working distance different wavelengths, often emitting several different objective is usually significantly more expensive and discrete wavelengths. The argon-ion laser has two may have a lower NA (Iower resolution) compared to main emissions, 488 nm (blue) and 514 nm. The a more conventional working distance lens. Kr/Ar laser has 3 main emissions at 488 nm (blue), Look Up Tables (LUT): computer algorithm for 568 nm (yellow) and 630 nm (red). adding colour to your images. Each grey level in the Lateral resolution: resolution in the plane offocus (x-y image is denoted a specific colour or shade of colour resolution). Lateral resolution is proportional to the for creating colour gradients. A dark green - green - inverse of the NA of the objective (i.e. high NA yellow - white gradient LUT can be used for objectives have significantly higher lateral resolution). displaying green fluorochromes such as FITC. Other Latex beads: small latex (plastic) spheres (also called LUTs can be used to delineate particular areas of latex microspheres or FluoSpheres) from 0.02 Ilm up interest, for example a LUT may be used to set the to 15 Ilm in diameter and stained with a range of correct dynamic range during collection by applying fluorescent dyes. Used to study endocytosis and discrete colour to pixels that are under- or over• intracellular movement. Also used as a very sensitive saturated. A LUT is applied to grey-scale images method of antigen detection by surface coating with a without the intensities being altered. The LUT suitable antibody. Sub-resolution latex beads can also coloured image can be converted to an RGB (Red, be used to measure the PFS (resolution) of the Green, Blue) colour image. microscope. Low contrast image: an image comprised of midrange Line averaging: each line is scanned several times, and grey tones with little or no black or white in the the average is displayed. This will increase both the image. sensitivity and the signal to noise ratio of the image. The advantage over screen averaging is that small 454 Glossary LP (Long Pass) optical filter: allows longer foeus light later as in a eonfoeal mieroseope. A multi• wavelength light to pass through and blocks the photon mieroseope is partieularly suited to relatively transmission of shorter wavelength light. deep penetration of whole tissue sampies, and ean be LSCM: Laser Seanning Confoeal mieroseopy. used to excite "UV" dyes such as DAPI as weil as LUT: see "Look Up Tables". visible wavelength dyes. Magenta: a light purple eolour formed when green light Multi-tracking: image eolleetion using laser line is subtraeted from white light (leaving red + blue). switehing to eolleet alternate ehannels with pre-set CMYK (Cyan, Magenta, Yellow and Blaek) inks are laser line excitation and eolleetion optics (for used in most printers. eliminating bleed-through between ehannels). High• Magnification: an image that is the result of an speed eleetronie switehing of laser lines ean be used to enlarged view of the objeet. An inerease in image size eolleet alternate image lines with different laser beyond the resolution of the mieroseope will result in settings. a larger image, without any inerease in resolution (see NA: see "Numerieal Aperture". "Empty magnifieation"). Historieally the Nanometre (nm): unit of length used to measure "magnifieation", denoted for example as 1000X was wavelengths of light. 10.9 meters. The Kr/Ar laser displayed along with the image. However, a more produces 488 nm (blue), 568 nm (yellow) and 647 nm accurate method of displaying the magnifieation is to (red) lines. use ascale bar within the image. In this way ehanges NB (Narrow Band) filter: allows a narrow range of in "magnifieation" due to the size at whieh the image wavelengths to pass through. For example a 605DF32 is printed or displayed do not affeet the denoted seale (diseriminating filter) is an optieal filter that allows a of the image. narrow band of light (32 nm) to pass, centred around a Median filter: image proeessing method that has the wavelength of 605 nm. property of smoothing the image (removing noise) ND (Neutral Density) filter: optieal filter that blocks while maintaining edges. This algorithm has a light of all wavelengths equally. Used to attenuate the tendeney to ereate "false" edges in the image and so amount of laser light reaehing the sampie. should be used with eare. Near Infrared (NIR): the light speetrurn from Megabyte (MB or Mbyte): 1,048,576 (220) bytes. approximately 750 to 2500 nm. Mercury lamp: powerful are lamp used as a light Nipkow disk: a disk with a spiral pattern of holes souree in normal fluoreseenee microseopy. The arranged so that, as the disk spins, light shining emission speetrum is from the UV to the far red. The through the disk seans every part of the specimen with Hg are lamp has a limited life span of approximately aseries of small points of light. A number of eonfoeal 200 hours, and should be ehanged at the time microseopes are based on the use of a Nipkow disk, reeommended by the manufaeturers. either alone or in eombination with a miero-Iens array Micro-Iens array: the Yokogawa Nipkow spinning disko disk eonfoeal mieroseope sean head also ineorporates Noise: in a eonfoeal mieroseope noise is due to both a miero-lens array disk that foeuses the irradiating optieal or shot (Poisson) noise (due to the statistical light through the pinholes in the Nipkow disk - thus nature of light, and of partieular eoneern when the inereasing the amount of light available at the sampie fluoreseenee intensity of the sampie is relatively low), for exciting fluoreseent dyes. and eleetronic noise of the instrument (this is usually Micron (Jl or Jlm): Ij.tm = 1000 nm (10.6 meters). The quite low, unless the gain level is set too elose to limit of resolution of light mieroseopy is 0.1 to 0.2j.tm maximum). Noise is manifested as "speekle" in the (100 to 200 nm), depending on the wavelength oflight image. and the NA ofthe objeetive. Nomarski optics: see "Differential Interferenee Multi-photon: exeitation of a fluorophore using long Contrast (DIC)" opties. wavelength infrared light to produee short wavelength Normal incidence: an angle of ineidenee of zero blue or green light. Also known as 2-photon and 3- degrees. photon exeitation. Multi-photon mieroseopes use very Numerical Aperture (NA): is the sine of the angle short pulses of high-intensity infrared light to exeite under whieh light enters the objeetive. This number fluoreseent probes. Only the foeus point has sufficient determines the amount of light an objeetive lens lets light intensity for multi-photon fluoreseenee to oeeur - through (the light gathering power of the lens) and is which results in only the foeal plane being visible, i.e. also related to the resolving power of the objeetive optieal seetioning is aehieved by seleetively exciting (the higher the NA the better the resolution). The the foeus plane rather than eliminating the out-of- Glossary 455 highest resolution lens generally available is an oil microwave heating are often used to permeabilise immersion 60x or 63x NA 1.4. fixed cell and tissue sampies. Streptolysin 0 or some Nyquist criteria: (also Nyquist sampling) pixel detergents can be used to permeabilise live cells. resolution should be set at half the separation of the Phase annulus: a ring shaped aperture placed in the optical resolution (this means an optical resolution of condenser to produce illumination for phase contrast 0.211m would require a pixel size of no large than microscopy. A specific phase annulus is required to O.ll1m). In practical terms you should aim for match the phase plate in the objective. approximately four pixels across the object you wish Phase contrast: uses the retardation of light by the to resolve. This means 16 pixels for 2D images (4 x 4 specimen to produce phase differences, which are pixels) and 64 pixels for 3D images (4 x 4 x 4 pixels). converted into contrast. This technique is used Objective: a complex system of lens components that extensively to image unstained live cells. However, produces most of the magnification, and its numerical phase contrast imaging is rarely used on the confocal aperture (NA) limits the resolution achievable. High microscope as contrast can be readily increased by NA lenses are very expensive, delicate and are altering the gain and blackness levels on the critically important for a good quality image. instrument, or alternatively very high quality Ocular: see "Eyepieces" transmission images can bc obtained using DIC optics. OG (Orange Glass): long pass colour absorption Phase plate: the glass plate placed at the back focal glasses that absorb more than 99.999% of light of plane of a phase objective that contains the phase ring. shorter wavelength energy (blue light). Phase ring: a darkened ring on the phase plate which on immersion objective: an objective lens designed (normally) retards light less than the phase plate. This for use with oils that have a refractive index equal to ring is used in phase contrast microscopy together that of glass (1.515). Effectively this means that the with the phase annulus to produce contrast from phase lens is in continuum with the coverslip. Resolution differences between light rays from different parts of and contrast is not affected by small movements of the the specimen and the background. objective (as when focusing on different parts of the Photobleaching (fading): loss of fluorescence from the specimen). area of the sampie that has been intensely irradiated Optical density: a logarithmic unit oftransmission. OD with light. Caused predominantly by the production of = -log (T). reactive free radicals. The rate of fading can be Optical sectioning (optical slices): the process of controlled by addition of antifade compounds (anti• recording images at different focus planes through the oxidants) such as n-propyl-gallate or p• specimen using a confocal or multi-photon phenylenediamine for fixed sampies, or Vitamin C microscope. A stack of optical sections can then be (sodium ascorbate) for live cells. The amount of reconstructed into a 3D representation ofthe object by bleaching is dependent on the fluorescent molecules using suitable software. used, the amount oflight used to illuminate the object, Parfocal: is where two or more objective lenses are in and the length of time the sampie is irradiated. focus at the same focus control position - particularly Photodynamic therapy (PDT): a cancer treatment useful when changing lenses. based on using photosensitising chemicals (specific PDF (Portable Document Format): Adobe's fluorophores) to kill cells that are exposed to light of a electronic publishing document format software for specific wavelength. Also called photoradiation Windows, Mac OS, UNIX®, and DOS. You can view therapy, phototherapy or photochemotherapy. PDF files using the free Acrobat ReaderE software. Photomultiplier tube (PMT): the photoelectric device PDF files can represent both vector and bitmap used to detect light by converting light into an electric graphics, and can contain electronic document search current that can be amplified. Each separate channel in and navigation features such as electronic links. PDF a confocal microscope has its own PMT tube, except documents are generated using the Adobe Acrobat in the case ofthe META channel where a multi-PMT software. array is used to detect the light. Peak emission or excitation wavelength: wavelength Photon counting: a pulse counting mode whereby the of maximum emissionlexcitation of a fluorophore. PMT circuitry is set to only count pulses over apreset This is the emissionlexcitation wavelength usually threshold. One pulse correlates with a single photon. give in tables of fluorophores. This method of image acquisition is only useful in Permeabilisation: cells must be permeabilised to allow relatively low light level images, and results in a large molecules such as antibodies to penetrate to the significant reduction in noise levels. cellular interior. Detergents, organic solvents or 456 Glossary Photon of light: a quantum of light, based on Planck's Polychroie: a dichroic beamsplitter that has multiple quantum theory of light. reflection bands and transmission regions. PIC (Bio-Rad): file fonnat for Bio-Rad confocal Primary colour: the colours red, green and blue images, not to be confused with other PIC fonnats. (RGB). These colours are denoted by specific Can be readily converted to many other formats using wavelengths within the electromagnetic spectrum and Confocal Assistant or LaserSharp. Many image are perceived as separate colours due to the method of processing programs can import Bio-Rad PIC files light detection in the human eye (the red, green and directly. blue colour cones). Computer screens generate the Pincushion distortion: a geometrical distortion of the large range of colours visible by simply mixing the image on the computer screen that makes a square amount of red, green and blue light present at each appear to bow inwards. On better quality monitors this pixel. pincushion distortion can be altered by using controls Primary image plane: the image plane where an image on the monitor. Pincushion effects can be quite severe of the specimen is first fonned. when photographing the screen directly - in which Projection: the process of displaying a series of optical case avoid rectangular or square line objects in the sections as a single composite image. The composite image. image can be "projected" in different ways, including Pinhole: see "Confocal aperture". "average view", "maximum intensity", or Pinholes: small breaks in the coating of an interference "topographical". filter. Pseudocolour: the colouring of a grey-scale image by Pixel: "Picture Element", is the smallest unit of a digital assigning specific grey levels to specific colours or image. Pixels may be described by their spatial or gradients of colour using a Colour Look Up Table temporal coordinates and intensity. (LUT). For example the grey-scale image collected Plan: or planar, refers to a lens that has been corrected from FITC fluorescence can be coloured with a colour so that the resultant image is in-focus across the whole gradient of dark green, green, yellow and white to field ofview. denote the degree of fluorescence intensity - but the Plan Apo: plan apochromatic objective lenses are image could just as easily be coloured blue through to highly corrected for colour (red, green, blue and UV), white! spherical aberrations (for green and red or more PSF: see "Point Spread Function". wavelengths) and flatness of field. Quantum Dots: are semiconductor nanocrystals that Plane polarised light: light oscillating in one plane can be excited by blue light (for example, 488 nm) and only. emit fluorescence in a narrow emission from green PMT: see "Photomultiplier tube". through to far red, depending on the size Point Spread Function (PSF): the three-dimensional (approximately 1Onm) and composition of the diffraction limited shape fonned by an objective lens quantum dot. Quantum dots can be coated with a in a light microscope. The PFS is determined both by protective outer layer, and then surface labelIed with a the lens and the media in which the sampie is variety of biological molecules, including Biotin and mounted. Streptavidin. Polar: anything that transmits light in one plane only. Quantum efficiency: the efficiency with which a They are often made of a stretched plastic film fluorophore converts absorbed light into emitted sandwiched between two layers of glass, which fluorescent light. transmits light oscillating in a plane parallel to the Quantum yield: the fraction of excited fluorophore stretch. molecules that emit a photon of light. Polarisation beam splitter: an optical filter that Quarter wave plate: retards light by a quarter of a deflects the polarised laser light to the sampie while at wavelength, producing circularly polarised light. the same time allowing the randornly polarised Quenching: the loss of fluorescence caused by a variety fluorescent light emanating from the sampie to pass of phenomena, particularly the loss of energy by through to the detectors. production ofheat. Anti-fluorophore antibodies can be Polariser: apolar placed in the incident light (usually used to quench fluorescence (useful for studying below the condenser) to produce plane polarised light. accessibility to externally added ligands). Polarisation: restriction of the orientation of the Raster: the sequential scanning pattern used in confocal vibration of the electromagnetic waves in light. When microscopy and video imaging. vibrations are restricted to one particular angle, the Ratiometric dye: a fluorescent dye with either two light is plane-polarised. different excitation or two different emission Glossary 457 wavelengths that change in fluorescence intensity, but the wavelength of light used (shorter wavelengths in opposite directions, on binding specific ions. results in higher resolution). Calculating the ratio of the fluorescent light emitted at RG (Red Glass): long pass colour absorption glass that the two wavelengths, or at one wavelength in the case absorbs more than 99.999% of shorter wavelength where two excitation wavelengths are used, can be energy. RG glasses absorb blue and green light (thus used to accurately establish the amount of fluorescent appearing red). change that is due to binding specific ions (for RGB (Red / Green / Blue): colours used on the example calcium) independent of the concentration of computer screen to generate all other colours. Also the dye. denotes an image file consisting of 3 images Rayleigh criteria: two objects are said to be resolved if representing the red, green and blue components of a their separation is such that their diffraction patterns multi-colour image. (Airy disks) show a detectable drop in intensity RLE compression: Run Length Encoded image between them. This somewhat arbitrary criterion was compression algorithm (used by Windows BMP file decided to be approximately 20% of the peak format). intensity, which corresponds to the first dark ring of ROI (Region OfInterest): a defined area (or several the Airy disko defined areas) of an image may be used to scan only Real image: an image that is located on the opposite that region of the sampie. The defined area may be a side of the lens from the object and can be projected cirele, square, line or a randomly drawn shape. onto a camera film or the retina ofyour eye. Saturation: see "Colour saturation" and "Dye Real time collection: fast collection of images showing saturation" . molecular fluxes (e.g. Ca2+) or movement of Scan head: the confocal rnicroscope scan head contains subcellular components within cells. The collected the scanning mirrors, and for most manufacturers the images can be displayed as a time lapse "movie". dichroic mirrors and sometimes the barrier filters. The Red, Green and Blue: see "RGB". scan head is mounted direct1y on to a conventional Reef Coral Fluorescent Protein: (RCFP) is aseries of light microscope. fluorescent proteins eloned from various reef coral Scan speed (scan rate): the speed of scanning the species (Discosoma sp) that can be readily attached by image. The final image scan rate is determined by the gene fusion to any protein of interest, allowing one to scan speed of the scanning mirrors, or in the case of a follow the fate of the protein in real time in living Nipkow disk instrument the speed of the spinning cells. Also see "Green Fluorescent Protein" (GFP). disk, and whether line averaging or binning has been Reflectance imaging: see "Backscatter". implemented. Refraction: the change in direction of oblique light Scanning mirrors: mirrors in the scan head that scan passing through a material of a different refractive the laser fast in the "x" direction and more slowly in index. See "Snell's law". the "y" direction. Refractive index (0 or R1): the ratio of the speed of Scanning zoom: areal increase in resolution (and propagation of light through a vacuum to that through magnification) can be obtained by zooming the the specimen. Vacuum = 1.0, glass = 1.5, water = scanned image (scanning a smaller area) in laser 1.333, immersion oil = 1.515. The direction of travel scanning confocal microscopy. When in zoom mode of light is altered (refracted) on moving from media the scan area can be moved by using the panning keys. with one refractive index to another. Screen averaging: screen averaging is used to collect Registration shift: a shift in the apparent position of the multiple screens and display them as an average (thus specimen when an optical element is inserted or lowering the noise level in the image). removed. Secondary colours: colours derived by mixing the three Resei: diffraction limited resolution limit of light primary colours. Red + blue = magenta (light purple), microscopy. This depends primarilyon the NA of the red + green = yellow, blue + green = cyan (light blue). lens. The resellimit for a 1.4 NA oil immersion lens is The secondary colours can also be thought of as the 0.1 to 0.2 Dm. "inverse" of the primary colours, for example, cyan is Resolution: the ability of an optical system to the full visible light spectrum without green. Most distinguish fine detail in a specimen (i.e. the ability to printers use the ink colours CMYK (Cyan, Magenta, image separately two neighbouring points of Yellow and Black). information). The resolution limit of an objective is Segmentation: see "Image segmentation". determined by the Numerical Aperture (NA) of the Semi-silvered mirror: a rnirror that reflects half the lens (the higher the NA the better the resolution) and incident light and transmits the rest. 458 Glossary Serial seetions: adjacent physical or optical sections of itself, the improper use of the objective, incorrect tissue or cells. immersion media or the sampie. In confocal SETCOL (Bio-Rad): Bio-Rad "Set Colour" Look Up microscopy spherical aberration is manifested as a Table (LUT) used to accurately set the upper and serious loss of light in the image or allocation of lower light levels (gain and black level) in the image. fluorescence to the wrong image plane, often in The lowest light levels are displayed green and the particular areas of the image or in the periphery of the highest light levels red, with a grey scale gradient in field ofview. between. An ideal image will contain a touch of green Spring LUT (Bio-Rad): Bio-Rad green Look Up Table and a touch of red with most of the image displayed as (LUT). The image is coloured with a gradient of dark shades of grey. green - light green - yellow - white. Sharpening: image-processing method used to enhance Standard tube-Iength optics: a fixed focal length of edges or other transitions in the image. This type of the objective (usually 160 mm). This is in contrast to image manipulation is often useful when producing a infinity-corrected objectives, where the light rays are slide for presentation, as "blurred" images are focussed to infinity (parallel light rays). Most modern particularly distracting for the audience. optical microscopes now use infinity corrected optics. Short Pass optical mter: see "SP" Stereology: a type of image analysis based on the Signal-to-noise ratio (S/N): is the ratio of the signal statistical analysis of numbers of objects, their size, coming from the sampie and the unwanted signal and orientation. caused by various optical and electronic components Stokes shift: the difference between the wavelength of within the microscope and associated electronics. An excitation and the wavelength of emission of a image with a higher signal to noise ratio is a better fluorophore. quality image. Three dimensional (3D) reconstruction: computer SUde maker: an instrument for making photographic software rendition of a 3D volume of interest slides from digital files. The instrument consists of a contained within a z-series of digital images. high-resolution grey-scale monitor that is Three-photon: see "Multi-photon". photographed using colour filter wheels to create a Thresholding: selection of regions of interest within an colour image. Image quality is better than that image based upon pixel grey level value. Thresholding obtained by the best colour monitors. Also known as a usually selects pixels above or below a single "film recorder". specified level, sometimes called "binary Smoothing: an image-processing method to remove thresholding". Greyscale thresholding, or "density high frequency noise, or widely variant pixel values. slicing", selects pixels lying within the limits of a This often has the effect of blurring the image, but is range of intensities. The specified grey levels are particularly useful for making noisy, or "grainy" usually replaced with asolid area of colour. images look much better. TIFF (Tagged-Image File Format): is used to SneU's law: light bends towards the normal (an exchange files between applications and computer imaginary reference line drawn perpendicular to the platforms. TIFF is a flexible bitrnap image format surface) as light passes from lower to higher refractive widely supported by virtually all paint, image editing, index material. and page-layout applications and does not alter image SP (Short Pass) optical mter: allows shorter intensities or colour table. Can be either compressed wavelength light to pass through and blocks the or uncompressed. transmission of longer wavelength light. Transmission ßuorescence: fluorescence microscopy Spectroßuorometer: an instrument for measuring the where the illuminating light is transmitted through the excitation (absorption) and emission spectra of condenser (as in conventional Bright-field ßuorescent molecules. microscopy) and the fluorescent light is collected by Spectrum: the visible light spectrum ranges from violet the objective. This type of fluorescence microscopy is light (400 nm) through green (500 nm) to red (700 quite dangerous for the user, as a correctly placed nm). Ultraviolet (less than 400 nm) and infrared barrier filter is critical for stopping the powerful (greater than 700 nm) are wavelengths of light often illuminating light (often UV) from entering the used in microscopy that are beyond the limits of the observers eyes (or the PMT tube used in confocal human eye to detect. microscopy). Transmission fluorescence is now only Spherical aberration: the inability of a lens to focus used in very specialised instruments. axial and marginal light rays to the same point. Transmission image: a transmission image of the Spherical aberration can be caused by the objective object in a confocal microscope can be obtained by Glossary 459 collecting the laser light that passes through the Water immersion objective: an objective lens condenser. A high quality transmission image of designed for use with water to form a continuum living cells can be obtained by collecting a very between the lens and the coverslip. Water immersion evenly lit low contrast image (Kaiman average if objectives may result in better resolution when possible) and then contrast stretching before saving. imaging water based biological sampies. Some The transmission image is NOT confocal (i.e. the immersion objectives have an adjustable collar to transmission image contains light from other focal allow them to be used with either water, glycerol or oil planes - it is not an optical slice). DIC and Phase as the immersion media. A water immersion objective contrast images can also be collected by using the is designed to be used with the correct thickness necessary optical components on the microscope. coverslip, unlike the water "dipping" objective where Tube length: the physical distance between the no coverslip is required. objective and the eyepiece. Wavelength oflight (A): The distance in nanometres Tungsten lamp: the lamp traditionally used for between nodes in the wavelength of light. Shorter conventional transmitted light microscopy. wavelength light has higher energy. The colour of the Two-photon: see "Multi-photon". light is determined by the wavelength. U1traviolet (UV): light from the region of the WB: "Wide Band" optical filters combine rectangular electromagnetic spectrum with wavelengths between band shapes with broad regions oftransmission. 100 and 400 nm (not visible to the human eye). UV White light: the combination of all three primary light is highly destructive to living tissue, and so must colours, red, green and blue. Illuminating all three be used with care on living cells. However, there are primary colours together produces white on a many excellent dyes available that require UV computer screen. However, white on a printed page is irradiation to generate fluorescence. Confocal produced by the absence of printing ink (displaying microscopes that use a UV laser are available, but they white paper). are difficult and expensive to run. UV A = 320 to 380 Wide-field: an epi-fluorescence microscope in which nm, and UVB = 280 to 320 nm. the full field ofview is illuminated. UV laser: UV lasers are very expensive, but do allow Wollaston prism: prism used in interference imaging one to use UV excited fluorophores such as Hoechst (see "Differential Interference Contrast (DIC)" dyes and various calcium indicators. Many UV dyes microscopy), consisting of a beam splitter made of can also be excited by using a multi-photon two wedges of a birefringent crystal such as quartz. microscope or the violet (405 nm) solid state laser The position ofthe Wollaston prism can be adjusted to now available. create a lighter or darker "shadowing" effect, which is Virtual image: a magnified image located on the same particularly valuable in live cell imaging. The side· of the lens as the object. The image cannot be Wollaston prism in some instruments may interfere projected onto film, but can be seen when looking with the collection of high-resolution fluorescence through the lens (an example of a virtual image is that images. seen when looking through a hand held magnifying Working distance: the distance between the front lens glass that is held relatively close to the specimen). of the objective and the coverslip. A long working Visible light spectrum: light from the region of the distance objective is very useful for microinjection, electro magnetic spectrum with wavelengths between but usually has a lower NA value (lower resolution 400 nm (blue) and 750 nm (red). and lower light gathering capacity) and is more Vital stain: a dye that is tolerated by living material. expensive compared to anormal short working Many fluorescent stains can be used as vital dyes as distance objective. long as a sufficiently low concentration of the dye is x-z scanning: the ability to collect an image in the X-Z used. Some dyes are excluded from living cells, but plane by scanning a single line at a series of focal will readily stain dead cells (the basis of the positions. commercially available LivelDead staining kits). Yellow: a colour that looks exactly the same whether Voxel: a three dimensional pixel (Volume Element) in a produced as a "pure" wavelength of light (around 560 digital image stack. nm), or a colour produced by the combination of red Water dipping objective: an objective designed to be and green light (600 and 500 nm), which is often used immersed into the culture dish from above (used for to denote regions of co-Iocalisation in a digital image. microinjection and electrophysiology). This type of Yellow is an important colour used in printing lens is usually made of a ceramic material and is used (CMYK; Cyan, Magenta, Yellow and Black) inks are without a coverslip. used in most printers. 460 Glossary Z-axis: the vertical axis in a microscope. Z-sections Z-resolution: the resolution in the "z" direction (at right refer to aseries of images collected by stepping the angles to the scan direction). The z-resolution on a microscope fine focus with a focus motor drive, or by confocal microscope is less than the x-y resolution, moving the microscope stage or objective lens with a and is about 0.5 to 0.8 ~m when using a high piezo electric stepper. resolution 1.4 NA 63x oil immersion objective (the x• Zip disk: a reasonably fast and relatively cheap disk y resolution in this case would be approximately 0.1 to with 100 or 250 MB capacity. The drive can be readily 0.2 ~m). moved to different computers by attachment though Z-series: aseries of 2D images encompassing a sampIe the printer parallel port or USB port. volume collected at intervals in the focal axis (z• Zoom: method of increasing the resolution (and stepping) by stepping the focus motor drive between magnification) of the confocal microscope (scanning acquisitions. zoom) or of making a digital image larger without an increase in resolution (digital zoom). Index Absorbance spectra. See Fluorescence, Bio-Rad. See Confocal microscope GlowUnder, 113, 129, 131 excitation spectra manufacturers Green, 15, 155 Acbromatic, 56 Bit depth (in images) in Confocal Assistant, 171 in LaserSharp, 377 Aequorea victoria (Jellyfish) fluorescent 8-bit, 7,114,146,178 Indexed colour, 156 12-bit, 7,114,146,178 proteins, 232, 233, 237 RGB colour images, 156 16-bit, 114, 146, 178 Airy disk, 50, 51, 52, 72, 73, 92,115, 116, SETCOL, 113, 129, 158, 181 24-bit colour, 156 117, 136 printer inks CMYK, 157 32-bit, 114 yellow, 128, 157, 246 Amira 3D. See Imaging software Black level, 19,45,60,91,113,114,158 Antifade reagents, 29, 112, 195, 199,207, yellow, in confocal microscopy, 180 Bleed-through, 13, 17,76,85,87,89, 136, Colour in imagesuman perception, 154 213,274,319 192,193,200,240,242 commercially available reagents, 198 Colourplates, 15,23,105,131,247,261 adjustment ofmixers (Bio-Rad), 363, 378, 381 DABCO,198 Coma,48 high-speed laser line switching (multi-tracking), for fixed cells, 196 242,388 COMOS software (Bio-Rad), 365 for live cell imaging, 198 spectral separation, 363, 388, 400 Components of a microseope, 53 may lower fluorescence intensity, 276 Compound microseope, 39 not needed with Alexa Fluor dyes, 208 Bovine serum albumin (BSA) as a blocking agent, 274 Computer projector, 182 n-propyl gallate, 198, 207, 274 as a lipid carrier, 224, 228, 315 p-phenylenediamine (PPD), 198 Computer sereen Trolux, 198,319 Box size. See Image box size I to I (100%) display, 133, 153, 173 Vitamin C (ascorbic acid), 197,198,319 Brefeldin A, 226 display resolution (pixels), 150, 151, 153 AOBS beam splitter, 73, 78, 89, 111,403, Bright-field imaging, 39, 41, 42, 45, 60, 109 dots per inch (dpi), 147 resolution, 126 404,405,406,409 Carl Zeiss. See Confocal microscope Condenser, 42, 43, 60 AOTF tuneable filter, 78, 89, 90, 103, 111, manufacturers Condenser iris, 42 112, 128,253,359,361,362,366,403, CCD eameras, 11,94, 100, 115, 124, 140, ConfocaI Assistant. See Imaging software 404,406,422,425,440,442 271,431 attachment port, 53, 61 Confoeal microscope Apocbromatic lens, 60 expense,29 Apoptosis, 231, 281, 282, 291, 311, 320, 340 Nipkow disk instruments, 5, 11,91, 92, 93, 94 Atto Bioscience, 99, 432, 433 hardware, 65-100 Arrhenius plot, 290 PerkinEImer, 99, 438, 440 laser scanning (LSCM), 4, 65, 66 Ascorbic acid (Vitamin C). See Antifade VisiTech International, 99, 442 layout, 4, 67 reagents Yokogawa scan head, 99, 435 multi-photon microseope, 95 Astigmatism, 49 on a fluorescence microseope, 29 Nipkow disk instruments, 2, 5, 11, 18,65,72, Attachment of cells. See Live cell and tissue ChemicaI reactions (light induced), 197 91,92,94,99,100,120,124,140,218,228, 321,335,355,421 imaging Cbromaphor Analysen. See Confocal Atto Biosciences CARV & Pathway HT, 431 Attenuation oflaser light, 89 microscope mannfacturers PerkinEImer Ultra VIEW, 438 Atto Bioscience. See Confocal microscope ChromatIe aberration, 48 VisiTech VT Eye & VoxCell Scan, 441 manufacturers correction optics, 56 Yokogawa Nipkow disk scan heads, 435 Autofluoreseenee,6, 138, 139, 198, 199, correction optics in condenser, 60 operating difficulties, 29 242,275,282,320,321 Coemcient ofvariation (noise in images), scan head attachment port, 61 fixative induced fluorescence, 199,267,270 135 stit scanning confocal microseope, 2, 65 reduce using red light, 211 Coherent (laser manufacturers), 96, 97 which instrument is best?, 100 to visualise cell structure, 199 CoUectIon ruters, 120-22 Confocal mieroseope mannfacturers, 98, Background fluorescence, 274 accurnulate, 110 355 Backscatter (reflectance) imaging, 5, 47, 70, averaging, 121 a table of major manufacturers, 99 92,107, 108, 142,313,375 collection to peak, 121 address and contact infonnation, 335 Atto Bloseienee, 11,92,99,118,335,430-31 Barrier filters. See Filters (optical) exponential filtering, 122 Kaiman, 121, 376 a table of AUo Bioscience instruments, 432 Beam splitter, 6, 58, 78, 89, 111,360 single frame collection, 120 CARV, 94, 432, 433, 434 AOBS beam splitter, 73, 78, 89,403,404,405, Co-Iocalisation, 52, 116,240,244,247 contact address, 431 406,409 Colour in Images, 128, 131, 154, 179 Pathway HT, 432 polarisation beam splitter, 73, 359, 360 Bio-Rad Cen Seience, 1,99,331,335,356-81 24-bit images (24-bit), 156 Bi-directional scanning, 120,359, 366,404, a table of Bio-Rad instruments, 357 eolaUf in merged images. 157 412, 422, 425 application notes, 257 calauf to optimise image collection, 129 Biochemistry of single cells, 3, 25, 239, 254, computer generation, 155 CellMap eonfoeat microscopes 351 generation on a computer screen, 154 CellMap IC, 357 Bioptechs Delta T dish, 308, 309, 310, 311 human perception, 154 CellMap ID, 357 contact address, 356 Look Up Tabi.. (LUT), 71,102,103,128,154, Bioptechs microscopy chambers, 305, 308, fluorescence spectra Internet site, 202 157,179,241 309,310,311,342,346 LaserSharp software, 359, 364, 366, 372, 377- Bioptechs objective heater, 296 Autumn, 155 colour plate examples, 15, 131 81 Bioptechs perfusion chamber, 310 GEOG, 129,131,156,179 MRC eODfoesl microscopes MRC-lOOO, 357, 365 GlowOver, 113, 129, 131, 155, 181 461 462 Index MRC-1024, 110,357,364-76,366 Fluoview 300, 421, 425, 427 ease ofuse, 28 backscatIer imaging (B I & T I filter Fluoview 500, 421, 424, 425 Inverted images, 114 blocks),375 Fluoview 1000, 81, 421, 422, 423 misuse,29 dual & tripie labeHing (Tl & T2A filter FVX, 421 Digital zoom, 127 blocks),373 multi-photon instrument, 422, 424, 425 Digitisation, 113 filter blocks, 369 MX50-CF,II,421 Direct (external) detectors for multi-photon live ceH imaging (BI & open filter blocks), eontaet address, 420 374 Fluoview software, 428 microscopy, 98 sean head, 367 speetral separation, 81 Display resolution, 148 MRC-I024 multi-photon, 357, 366 Optlscan Imaging, 71, 73, 91, 313 DMSO, 267, 271, 315, 316 MRC-500, 357, 365 PerkInEImer, 11,92,99,118,140,321,336, Duallabelling. See multi-Iabelling MRC-600, 357, 365 437-39 Dvorak-Stotler perfusion chamber, 306, 307 Radlance instruments, 66, 67, 72, 73, 79, 103, eontaet address, 438 Edge enhancement filter. See Image 116, 358-M, 3511-M Ultra VIEW LCI, 439 processing filters Instrument Control Unit (ICU), 358, 362 Quorum Technologies, 336, 442 Miero Radianee, 357 Solamere Technology Group, 336, 442 Eight-bit images (8-bit), 7,114,146,178 Radianee 2100, 357, 359 VisiTech International, 11,92,99,140,321, Emission fingerprinting, 240, 241, 400, 401 Radianee 2100 Rainbow, 85, 86, 357, 362 336,439-41 Emission fmgerprinting (on-line), 400 Radiance multi-photon, 95, 98, 357, 359 eontaet address, 441 Emission spectra. See Fluorescence, emission Radianee scan head, 360 VoxCeH Scan,442 spectra sean rates, 118 VT-Eye,442 Endocytosis, 292 Signal Enhaneing Lens (SELS), 116,350, Vlsltron Systems, 337, 442 Enzyme activation energy, 291 359,364 Yokogawa, 11,92,99,140,321,337,435-37 RTS video rate confocal microseope, 357 contaet address, 435 Epi-fiuorescence microscopy, 6, 261 slit scanning confocal microscope. 66 CSUIO Sean Head, 435 Epi-illumination, 7 spectral separation, 81 CSU21 Sean Head, 437 ER probes. See Fluorescent probes, organelle SpectraSharp software, 364 Confocal microscopy probes trouble shooting Bio-Rad instruments, 376 advantages, I, 27 Excitation and emission spectra, 190, 191 Carl Zeiss, 1,99,331,335,381-401 applieations, 26 Excitation ofa fluorophore, 188 tonfoeal microscopes different types, 2 multi-photon exeitation, 189 a table of Zeiss instruments, 383 first instruments, 2 single-photon exeitation, 188 LSM 10/44, 383 history, 2 Excitation spectra. See Fluorescence, LSM 410/310, 383 image formation, 7 excitation spectra LSM 5 PASCAL, 383 international mailing list, 203, 326, 327 LSM 510, 383 irradiation of the sampie, 70 Exponential filtering. See Collection filters, LSM 510 META, 68, 81, 383, 384-90 limitations, 27, 29 coofocal images META Channel, 388 original patent (Marvin Minsky 1957), 2 Extended focus image, 6, 20, 21, \07, \3 7, sean head, 386 out of foeus light removal, 9 138 LSM 510 NLO multi-photon, 383 prineiples, I External (direct) detectors for multi-photon ConfoCor 2 (FCS), 383 raster pattern, 8 microscopy,98 eontaet address, 382 sensitivity, 27 Extinction coefficient. See Fluorescence, speetral separation, 81 skills required, 3 Zeiss LSM software, 391-95 UV laser mieroseopy, 88 extinction coefficient z-stepper motor, 61 what is it, 1-29 Eyepieces (oculars), 6, 39, S8 Chromaphor Analysen-Technik, 335, 442 whyuse it?, 27 Fast (xZ) & Fastest (x4) scanning (Bio-Rad), Lelca Microsystems, 1,99,332,335,402-14 Coofocal principle, 9 \03,379 confocal microscopes Constructive interference (light), 33, 34 FastX-Y (Zeiss), 103,393 a table of Leica instrument, 403 Contrast stretched images, 158 Fibreoptic ICM 1000,403 eonfocal pinhole, 73 spectral scanning instruments, 81 Control panel (programrnable), 91 Converting Bio-Rad PIC images, 169 connection to sean head, 66, 71, 79, 103,359, TCS SL, 403 384,402,415,417 TCS SP2 AOBS, 78, 81, 403 Cora! (Discosoma sp) fiuorescent proteins, laser eonneetion, 87, 89, 386, 404, 421, 422, 425, sean head, 405 234,238 437,439,442 TCS SP2 MP multi-photon, 403 CoreDRAW. See Imaging software miniaturised confocal endomicroscope, 73 TCS SP2 RS (resonant scanner), 118,403 Coverslip thickness, 44, 56 multi-photon laser eonnection, 96 contact address, 402 transmission piekup, 53, 60, 62, 108, 359, 366 heated microscope enclosure, 297 Curvature of Field, 49 Leiea software, 408-14 Cytodex (dextran) beads for growing cells, ii, Field aperture, 42, 60 multi-photon microscope, 407 22 Flle formats, 147, 162 sean rates, 118 CytospinD centrifuge, 268, 285 BMP, 162, 168, 169 GIF,162, 168, 169 spectral separation, 81 DAß (diaminobenzidine), 259 JPG (JPEG), 147,162,168,169 McBain Instruments, 336, 442 DAßCO. See Antifade reagents Meridian Instruments, 66 PIC (Bio-Rad), 143,162, 165, 166, 168, 169, 171, Dagan imaging chamber, 30S, 333, 345 sUt scanning confocal microseope. 66 173,331,377 Nlkon Instruments, 99, 336, 414-19 Data backup, 144 TIF,I56,162,I68, 169,171, 173 confocal microscopes Deconvolution, I, 166 File names, 142 a table of Nikon instruments, 416 Destructive interference (light), 33 Filter separation of fiuorescent light, 73 CI Digital Eelipse, 103,416 DIC (Nomarskl) imaging, 46, 109,390,407 Filter wheel spectral separation, 86 sean head & deteetor unit, 418 from bright-field images using QPm (Iatia), 47 Filters. also see collection and image PCM 2000, 416 Dichroic mirrors, 71, 79, 360, 361, 363, 369 processing filters RCM 8000 high-speed eonfoeal, 416 560 DCLP long pass mirror, 70, 75 Nikon C I software, 419 Fllters (optical), 60, 71, 79, 362, 363, 369 Tl tripie dichroie, 70, 75 E600LP long pass filter, 70, 77 Nikon eontaet address, 415, 420 T2A 560 long pass mirror, 75 Olympus, 99, 332, 336 filter terrninology, 79 what is a diehroie mirror, 74 HQ5 I 5/30 band pass filter, 70, 77, 79, 80 confocal microscopes Diffraction oflight, 7, 33, 34 a table ofOlympus instruments, 421 interference filters, 76 Digital images, 14~2 long pass filter, 77 Index 463 narrow band filter, 77 Alexa Fluor 680, 209 duallabelling, 195,206,241 orange glass (00), 77 Alexa Fluor 700, 209 excitation & emission maxima, 207 supply companies, 342 Alexa Fluor 750, 209 filter blocks for fluorescein, 75, 77, 79 what is a barrier filter, 76 fluorescence spectra, 208 fluorescence spectra, 194, 206 Finding YOUf sarnple, 104 spectra, 208 immunolabelling, 208 FISH, 239, 256 aminocoumarin, 205 pH dependency, 207 Fixation, 267 APC-Cy7 conjugates, 205 Green FIuorescent Protein (GFP), 232--38, 316 a table of common derivatives, 237 aldehyde fixatives, 269, 275 apoptosis markers PhiPhiLux, 230 applications, 235 formaldehyde, 269, 270 coloured variants, 233 glutaraldehyde, 267, 270, 275 BODIP~,205 Calcein, 220, 230 (Discosoma sp) paraformaldehyde, 270 Coral fluorescent proteins, 234, commercial fixative preparations, 270 calcium indicators, 218-20 238 a table of common calcium dyes, 220 enhanced derivatives, 233 cryo-sectioning, 271 fixative induced fluorescence. See BTC,220 excitation light, 234 Autofluorescence Calcium Crimson, 220 Jellytish (Aequorea vicloria) fluorescent Calcium Green, 220 microwave fixation, 270 proteins, 232 Calcium Orange, 220 limitations, 236 organic soIvents BS fixatives, 268 Fluo-3, 219, 220 molecular associations, 25 acetone, 269 acetone/methanol, 261, 268, 269 Fluo-4, 220 Sea Pansy (Renilla reniformis) fluorescent methanol, 268 Fura Red, 219, 220 proteins, 233, 238 paraffin embedding, 271 Fura-2, 218, 219, 220 spectra ofGFP and derivatives, 236 Indo-I, 218, 220 permeabilisation, 270 structure, 232 picric acid, 270 Oregon green BAPTA, 220 suppliers, 340 Quin-I,220 hydroxycoumarin, 205 FLASH - hexa-peptide fluorophore tag, 238 Rhod-2,220 ion indicators, 216, 222 FLlM,255 X-Rhod-I,220 also see calcium indicators. FLlP,252,253 Cascade Blue, 205 copper indicators, 222 Flow eytometry, 204 cell integrlty prob.. Mag-fluo-4,220 Fluar lens, 56 Calcein, 230 Mag-indo-I, 220 F1uorescence cell tracers, 228, 230 Magnesium Green, 220 best excitation wavelength. 192 CellTracker, 228, 230 nitric oxide indicators. 222 brightness of a fluorophore, 191 CMFDA, 228, 230 latex beads (microspheres), 131,231,322 changes to excitation & emission spectra on fluorescein diacetate, 230 Lissamine rhodamlne, 195,205,207,209,264 binding proteins, 191 Green Fluorescent Protein (GFP), 228 excitation & emission maxima, 207 emission spectra, 190 latex beads (microspheres), 230 fluorescence spectra, 207 environmental influence on fluorescence spectra. Lucifer yellow, 230 Lucifer yellow, 250, 279 191 membrane probes as cell tracers, 230 membrane potential probes, 222 excitation (absorbance) spectra, 190 cyanine (Cy) dyes also see mitochondrial probes. extinction coefficient. 190, 191 a table ofexcitation & emission maxima, 211 oxanol,222 fluorescence spectra (reading correctly), 192 Cy2, 205, 210, 211 membrane prob.. , 227, 229 image collection, 102 Cy3, 205, 210, 211 bis-pyrene-PC, 227, 229 imaging, 13,47 Cy3.5,211 BODlPY®-cerarnide, 227, 229 immunolabelling, 259-77 Cy5, 205, 210, 211, 267 C,rfluorescein, 229 multi-photon fluorescence, 189 Cy5.5,211 C4-BODlP~ C9, 229 peak wavelength of absorption, 189 Cy7,211 cis-parinaric acid. 229 quantum yield, 190, 192 Cyanine dyes, 210 DiA,229 quenching, 265 DiI/DiO. See membrane probes Dil,36, 122, 134,227,229 single-photon (conventional) fluorescence, 188 disruption of cells, 29 DiI/DiO,315 spectra, 202 DNA probes, 212 DiO, 117,229 Stokes shift, 190 7-AAD (7-arninoactinomycin D), 215 DPH, 227 what is it?, 185-200 a table of DNA probes, 215 FM 1-43,229 Fluoreseenee Resonance Energy Transfer, acridine homodimer, 215 FM 4-64,229 acridine orange, 212, 215 NBD-C.-HPC, 229 SeeFRET APOPTRAK,215 N-Rh-PE, 227, 229 Fluoreseenee Speetra Internet Site (Bio-Rad), cell-permeant dyes, 215 methoxycoumarin, 205 203 cyanine dimers (TOTO, YOYO, BOBO etc), molecular structure, 194 Fluorescent cloth autofluoreseenee (test 215 NBD,205 sarnple), 6, 138, 139 cyanine monomers (TO-PRO, LO-PRO, BO- organelle prob.. , 223-26 Fluoreseent plastic (test sarnple), 141 PRO etc), 215 a table of organelle probes, 224 F1uorescent probes, 201-38 DAPI, 87, 96, 212, 214, 215 ER,225 dihydroetbidium, 212, 215 Brefeldin A, 226 acridine orange (also see DNA probes), 10, 140, DRAQ5,215 Dil,225 177,191,212 ethidium bromide, 5,145,214,215 DiO, 225 Alexa Fluor dyes, 193, 195, 204, 207, 208, 209, 214,265,319 Hoechst, 87, 96, 214, 215 DiOC., 226 propidium iodide, 15,22,193,213,215,231, ER-Tracker Blue-White DPX, 225 a table of absorbance & emission maxima. 209 247,261,263 fluorescent Brefeldin A, 225 Alexa Fluor 350, 209 Alexa Fluor 430, 209 SYTO (cell permeant) stains, 212, 214, 215 rhodamine B hexyl esters, 225, 226 Alexa Fluor 488, 205, 208, 209, 264 SYTOX (dead cell) stains, 212, 214, 215 Golgi, 224 Alexa Fluor 532, 205, 209 TOTO,214 BODlPY' C.-ceramide, 224, 226 Alexa Fluor 546, 205, 208, 209 UV dyes, 96, 212, 215 NBD C.-ceramide, 224, 226 Alexa Fluor 555, 209 YOYO, 214, 215 lysosomal, 225, 226 Alexa Fluor 568, 205, 208, 209 esterase derivatives, 217 fluorescent dextran, 225, 226 Alexa Fluor 594, 205, 208, 209 Ouorescein (F1TC), 71, 128, 190, 194, 195, 205, latex beads (microspheres), 225 206, 210, 264, 267 LysoSensor, 221, 222, 225 Alexa Fluor 647, 209 LysoTracker, 221, 225, 226 Alexa Fluor 660, 209 a table oftraditional fluorescent probes, 207 conjugation to antibodies, 207 mitocbondrla, 223, 224 464 Index JCI, 191,224,225 Healed enclosure (microseope). See Live cell Lucifer yellow, 250, 279 JC9,224 -and lissue imaging malariaparasites, 5, 10,15,131,140,145,154, MitoTracker, 263, 282 Healed microscope stage. See Live cell and 177,247,250,261,279 MitoTracker Green, 102, 105, 168,223,224 merged images, 15,22,105,247,250,261,279 MitoTracker Red, 102, 105,223, 224 tissue imaging, lemp conlrol MitoTracker Red/Green, 102, 105, 168 MitoTracker Yellow, 223 High speed image collection. See Image MOLT-4 cells, 20, 21, 123, 125, 127, ISO, 153, nonyl-acridine orange, 224 collection, high speed 158,322 rhodamine 123,223,224 Hislogram (of image intensity values), 160, monitor display resolution, 153 tetramethylrhodamine (TMR), 223, 224 161 NBD-ceramide, 21,123,125,127,158 PE-Cy5 conjugates, 205 Honesty In ImagIng research, 177-80 optical slices z-series, 20, 107, 138 PE-Cy7 conjugates, 205 difficulties when presenting single images, 179 pig skin (backscatter imaging), 107 PE-Texas red, 205 honest presentation of images, 177 Poisson noise, 36 pH indieators, zn Iatia (QPm software), 47 propidium iodide, 15,22,247,261 a table of common pH indicators, 221 red blood cells, 18, 252 BCECF,221 IF A (Immunofluorescence Anlibody Assay), screen vs line averaging, 122 CFDA,221 259 single optical slice, 5,10,15,18,21,36,102, FDA,221 Image coUection, 101-44 105,109,117,122,123,125,127,131,134, HPTS,221 I to I image collection box size, 123 140,145,150,153,158,161,168,177,243, Oregon green, 221 512 x 512 collection box size, 123 247,250,261,279,322 SNAFL, 221, 222 collect grey-scale images, 181 stereo pair image, 22, 139 SNARF,221,222 collecting for publication, 181 surface label, 18,20,21,150,153 PhiPhilux, Z30, 340 collection box size, 1Z2-24, 123 surfaced labelIed red blood cells, 18 propidium iodide, 213 collection modes, 102 time series, 131, 140, 177 quantum dots, 230, 231 high-speed cOllection, 100,321 transferrin endocytosis, 15,20,150,153 quinine, 194 single-Iine cOllection, 323 transmission, 109, 250, 279 reactive fluorophores (table), 205 slow scan image cOllection, 323 transmission + fluorescence, 250, 279 Red 613,205 Image enhancing fillers, 159 Imaging chambers. See Live cell and lissue rhodamine, 128 Image formation, 8, 38,41 imaging rhodamine red-X, 205 Image histogram, 160, 161 Imaging depth, 315 R-Phycoerythrin (PE), 205 Image J. See Imaging software suppliers, 339 Imaging live cells. See Live cell and lissue tetrametbylrbodamine (TMR), 192, 193, 195, Image manipulalion, 158 imaging 196,205,206,207,208,210,264,267 Image pixels, 149 Imaging software, 163-76, 163, 164, 165 a table of traditional fluorescent probes, 207 Image prousslng ruten 2D and 3D image manipulation, 166 duallabelling, 241 edge enhancement, 159 a table ofprograms, 165 excitation & emission maxima, 207 Local Area Contrast, 159 Amira,I66 filter blocks fortetramethylrhodamine, 75, 77, median filter, 159 AutoDeblur, 166 79 sharpening filter, 159 AutoVisualize, 167 fluorescence spectra, 206 smoothing filter, 159 Confoeal Assistant, 15, 141, 161, 162, 164, 165, Te... red, 202, 205, 206, 207, 264 Images 168-72,173,174,175,331,359,366,377 excitation & emission maxima, 207 acridine orange, 10, 140, 177 converting PIC files, 169 filter blocks for Texas red, 75, 77, 79 autofluorescence, 6, 138, 139 image menu, 171 fluorescence spectra, 206 backscatter (refleetance), 107 main menu, 168 toxicity, 282 colourplate,I5,23,I05,I31,247,261 menu bar and tool bar, 170 TruRed,205 confocal (scanning) zoom, 125, 134 series menu, 172 Fluorescenl solution (tesl sample), 141 confocal pinhole adjustment, 117 Corel PHOTO-PAINT, 166, 175 Fluorile. See Objeclive lens, FLUAR contrast stretched, 158 CoreIDRAW, 164, 166,173, 175-76 for presentation and publication, 166, 167 F1uoview. See Confocal microscope cytodex (dextran) beads, ii, 22 dendritic cells in human foreskin, 22 GIMP, 165 manufacturers, Olympus DIe transmission, 109 GraphicConverter, 165 Flyback blanking, 120 digital zoom, 127, 145, 150 IDL,I67 Focus controls, 61 DiIlDiO, 36, 117, 122, 134 image collection software, 164 FRAP, 25,87,100, 112, 141,234,235,239, duallabelling, 15,22, 102, 105,243,247 Image Examiner (Carl Zeiss), 165,391 252,253,351 ethidium bromide, ii, 5,145 Image J, 110, 141, 161, 164, 166 FRET, 25, 87, 88, 205, 233, 234, 235, 237, extended focus image, 6, 20, 21, 107, 138 Image Pro Plus, 167 238,239,254,255,332,351 fixed cells, 5,15,18,22,145,161,243,247,261 Imaris, 167 fluorescent cloth, 6, 138, 139 IPLab,I67 Gain level (PMT Voltage), 113, 114 FRAP time series, 252 Irfanview, 165 GEOG. See Colour in images, Look Up H-ferritin endocytosis, 261 LaserPix, 167 Tables (LUT) high-speed imaging, 322 LaserSharp (Bio-Rad), 377--111 GlowOverfUnder. See Colour in images, HL-60 cells, 36,109,117,122,134,168 LaserSharp Image Analysis (Bio-Rad), 165 Look Up Tables (LUT) human skeletal museie, 161,247 Leica Lite (Leica Microsystems), 165,408 G1ycerol mountanl, 274 image collection box size, 123 MetaMorph, 167 Microview, 165 Golgi probes. See Fluorescenl probes, immunolabelled Band 3, 18 immunolabelled malarial parasites - FEST, 15 NIH Image, 110, 141, 161, 164, 166 organelle probes immunolabelled malarial parasites - QF 120, 15 Object Image, 161, 164, 166 Green Fluorescenl Prolein. See under immunolabelling, ii, 15, 18,20,22,161,243, Open/ab, 167 Fluorescenl probes 247,252,261 Photoshop, 109, 110, 128, 151, 158, 160, 161, Grey levels in images, 146, 178, 179 internallabei, 10, 15,20,21,36,117,122,123, 164,166,167,171,173-74,173,175,176, Grey-scale, 146 125, 127, 131, 134, 140, 168, 177,247,250, 182 Grey-scale transmission images, 249 261,279,322 channel merging, 250, 251 image display sereen, 173 Growing cells on coverslips. See Live cell latex beads (microspheres), 131, 322 live cells, ii, 10,20,21,22,36,102, lOS, 107, layer merging, 249 and lissue imaging, attach. of cells 109,117,122,123,125,127,131,134,140, plugin for Bio-Rad PIC files, 166, 173,331, Hair autofluorescence (Iesl sampie), 142 150, 153, 158, 168, 177, 252, 279, 322 377 Halogen lamp, 63 live human heart muscle, 102, 105 ,ele,ence books, 353 Index 465 tool bar, 174 Lab-Tek microscopy chambers, 286, 287, Intracel, 305 PowerPoint, 141, 166, 175,176 303,333 Ludin chamber, 306 QPm (Iatia), 47 Laser intensily at lhe sample, 110 simple flow chamber (VitroCom), 288 Quicktime, 165 supply companies, 346 Lasers (Uled In eonfoeal mieroseopy), 87 Scion Image, 141, 161, 164, 166 VitroCom, 288 a table of common lasers, 88 Slidebook, 167 quartz walled chamber, 288 argon-ion, 87, 88, 90 SoftWoRx, 167 simple imagIng ebambers, 282-118 excitation of fluorophores, 188, 194, 204, 206, Volocity, 167 concave weil microscope slide, 283, 284 208,213,219 VoxBlast, 164, 167 gasket chamber, 284 attenuation of, 89 Voxel View, 167 Lab-Tek dishes, 286, 301, 333, 343 damage to cells, 29, 320 Xnview, 165 MatTek dishes, 287, 301, 302 green-Iaser- Ludin perfusion chamber, 306 fluorescein excitation, 207 Optical aberrations caused by the specimen, LUT. See Colour in images, Look Up Tables fluorophore excitation, 95, 187, 188, 189 49 (LUT) GFP excitation, 234 Optical fibres, 73 hardware, 2, 65, 66, 95 Optical filters. See Filters (optical) Lysosomal probes. See Fluorescent probes, Hoechst excitation, 215 organelle probes imaging depth, 13,315 Optical sectioning, 19 Malaria parasites (images), 5, 10, 15, 131, imaging live cells, 25, 323 Optical slice, 10 140, 154, 177,247,250,261,279 imaging live tissue, 25 Optical slices z-series, 20,107,138 MatTek microscopy dishes, 287, 302, 333, inherent optical slice ability, 72 Optical slicing, 10 343 instruments, 99 Optimal image collection, 132-34 McBain Instruments. See Confocal international mailing list, 327 Optiscan. See Confocal microscope lack of exeitation of Cy dyes, 205 microscope manufacturers lasers, 88, 96, See separate index enlry manufacturers Median filter. See Image processing filters limitations, 29 Organelle identification, 26 Membrane phase transition, 291, 292, 293, loca1ised heating from laser, 196 OS/2 operating system, 364, 366, 372, 377 314 NADH and NADPH exeitation, 198, 199 Out offocus light, 4 Mercury arc lamp, 63 no need for pinhole, 72, 189 Over sampling, 126 Merged dual channel fluorescence image, photo dam.ge, 319 Panning to move around the sampie, 126 105 the Internet, 202 PAP pen, 272, 273, 274 UV dyes, 12,96,194,212,214,215 Perfusion chambers. See Live cell and tissue Merging fluorescence and transmission why use it? 27 images, 249, 250, 251 Multi-PMT array, 244 imaging META detection channel (Zeiss), 68, 81, 82, Multi-tracking, 242, 244, 388 PerkinElmer (Wallac). See Confocal 84,91,102,240,242,244,266,363,382, NAOH & NADPH, 198,320 microscope manufacturers 383,384,386,387,388,389,390,396, Nail polish, 197, 198, 274, 275, 276, 283, Petri dish warming plates. See Live cell and 397,400 284,286 tissue imaging Metal disk imaging chambers, 295 Necrosis,281 PFS (point spread function), 52 Methyl ester derivatives, 223, 316 Neutral density filters, 90, 112 pH control. See Live cell and tissue imaging Microinjection, 316 NeutrAvidin, 267 pH imaging, 26 Micro-Iens array, 94, 355 NIH Image. See lmaging software Phase contrast imaging, 46 Microscope attachment ports, 61 Nikon. See Confocal microscope Phenylenediamine (PPO). See Antifade Microscope care and maintenance, 55, 59 manufacturers reagents Microscope imaging chambers. See Live cell Nipkow disk, I, 11,72,92 Phosphorescence, 197 and tissue imaging extended time collection, 140 Photo darnage, 319 Microscope stage, 61 miero-Iens array, 93 Photobleaching,29, 126, 190, 195, 196, Microscopy,31--63 pinhole pattern, 92 197, 198,200,204,207,208,233,253, Microwave fixation. See Fixation, Noise in an image, 135 263,319 microwave N-PLAN lens, 56 Fixed cells, 196 Mitochondria probes. See Fluorescent n-Propyl gallate. See Antifade reagents Live cells, 197 probes, organelle probes Numerical aperture (NA), 42 Photodiode light detector, 91 Molecular Oynamics. See Confocal Nyquist sampIing, 93, 125, 126, 133 Photomultiplier tube (PMT), 5, 91,136 microscope manufacturers !imitations with Nipkow disk instruments, 93, 100 Photon counting, 37, 110, 363, 364 Molecular Probes, 202, 332 Objective heater, 296 Photoshop. See Imaging software Moss spores (test sampie), 142 Objective lens, 56 Physitemp Industries warming plates, 302 MRC-600, 1000, 1024. See Confocal achromat, 58 PID temperature controller. See Live cell and microscope manufacturers air (non-immersion), 44,58,317 tissue imaging apochromat, 56, 58, 60 Pinhole (confocal), 4, 6,7,37,68,71 Multi-labeUing (including duallabeUing), best lens for live cell imaging, 317 26,192,195,240,241,247 Airy disk, 116 cleaning, 59 in dual & tripie labelling. 116 choicc of fluorophore, 240 dipping objeetive lens, 58, 296, 318 large pinhole size, 116 choosing thc correet probes, 240 Fluar lens, 56 Nipkow disk pinholes, 11, 92 correct level of cell labeJling, 240 fluorite, 58 one Airy disk, 115 direct immunolabelling, 264 glycerol immersion, 44, 58 open pinhoie, 73,115 dual channel fluorescence imaging, 102 heater,296 optimal size, 115-17, 115, 117 duallabelling, 5, 15, 17,52,79,80,87,89, 116, infinity corrected, 56 physical iris within scan head, 72 180,243,266 magnification, 57 using fibreoptics, 73 image alignment, 28 multi-immersion, 57 variable pinhole, 72 image calouf designation, 128, 157 oil immersion, 44, 56, 58, 284, 296, 314, 315,317 very small size pinhole. 116 immunolabelling, 261 plan-apochromat, 56, 58 Pixels, 145, 146 minimising bleed-through, 242 water immersion, 44, 58, 296, 314, 315, 317, 318, PL (plan-apochromat) lens, 56 multi-channel collection, 241 376 multi-tracking,388 Offset control, 113, 114 Plastic coverslips, 289 sequential image collection, 245 Offset stereo pair image, 22, 139 PMT voltage (gain level), 113 tripie labelling, 5, 116 Point Spread Function (PSF), 51 Multi-photon microscopy, 1,5,12,17,31, Oil immersion Objective lens. See Objective lens Poisson noise, 10, 36, 37, 118 65,100,101,188 Poisson statistics, 136 advantages, 100 Olympus. See Confocal microscope manufacturers Polarisation beamsplitter, 73, 360 Bio-Rad Radianee 2100 MP, 95 Pollen grains (test sampie), 142 calcium green excitation, 220 One Airy disk, 117, Also see Pinhole DAPI exeitation, 215 (confocal) Poly-L-Iysine. See Live cell and tissue diffieuity in exeiting Cy dyes, 210 On-line emission fingerprinting, 400 imaging, attachment of cells direet (extemal) deteetors. 98, 406 Optical aberrations, 47 PowerPoint. See Imaging software excitation spectra, 191 PowerPoint presentations, 182 Index 467 Printers Sensitivity, I, 112 3D information from 20 images, 18,28 a table of printers, 185 SETCOL. See Colour in images, Look Up 3D reconstruction, 267 black and white laser printer, 184 Tables (LUT) 3D representation by rocking motion image, 137 extended focus image, 6, 20, 21, 107, 13 7, 138 colour laser printer, 184 Sharpening filter. See Image processing dye sublimation printer, 184 image collection, 136 filters inkjet printers, 184 Time-Iapse imaging, 17, 131,139-41,140, photographie paper, 185 Signal Enhancing Lens (SELS), Bio-Rad, 72, 177 Printing and publication, 180, 183 116,359,364 Tissue slices, 259 Protein trafficking, 26 Signal to Noise (SIN) ratio, 135 Titanium sapphire multi-photon laser, 96, 97 Publication of images, 177--85 Silanes. See Live cell and tissue imaging, Transfection, 316 Pulsed infrared lasers (multi-photon attachment of cells Transmission image, 91, 109 mieroscopy), 96 Simple lens, 38 TransmIssion Imaging, 5, 26, 45, 108-10, Quantitative analysis, 178 Single channel, 5 249,261,390,407 Quantitative analysis of digital images, 113, Single frame capture mode, 120 combined with fluorescence, 15 160, 180 Single labelling, 15 DIC images using QPm (Iatia) software, 47 Quantitative confocal microscopy, 256 Single photon excitation, 188 real eolour transmission imaging, 110 Quantitative fluorescence imaging, 28, 180 Single-line image collection. See Image Tripie labelling. See Multiple labelling Quantum dots, 230, 231, 340 collection, single-line Trolux (Vitamin E). See Antifade reagents Quantum yield, 192, 200 Sixteen-bit images (l6-bit), 114, 146, 178 Tungsten lamp, 63 Quenching (fluorescence), 265 Skirn milk (as a blocking agent), 274 Twelve-bit images (12-bit), 7, 114, 146, 178 Quinine, 194 Slide making, 181 Two-Photon microscopy. See Multi-photon Quorum Technologies. See Confocal Slit scanuing confocal microscope, 2, 65 mieroscopy microscope manufacturers Siow scan image collection. See Image Ultrafast pulsed infrared lasers, 97 Radiance. See Confocal microscope collection, slow scan Under sampling, 126 manufacturers, Bio-Rad Smoothing filter. See Image processing Upright microscope, 53 Reactive Fluorophores, 204 filters UV DNA dyes, 214 Red/green stereo pair, 139 Sodium ascorbate (Vitamin C). See Antifade UV imaging, 320 Reflectance imaging. See Backscatter reagents V ALAP (microscope slide sealant), 197,274, imaging Sodium borohydride, 270, 275 275,276,283 Refraction, 35 Solamere. See Confocal mieroscope Vibration, 68 Refractive Index, 35 manufacturers Vibration damping, 376 Region ofInterest (ROI) Scanning, 128 Spectra Physics (laser manufacturers), 96, 97 Vibration free imaging, 62 Renilla reniformis (Sea Pansy) fluorescent Spectral Prism, 404 Visible light spectrum, 187 proteins, 233 Spectral separation of ßuoreseent light, 73, VisiTech International. See Confocal Resolution, 1,4,5,50,73,267,280,282, 8HI6,241,266 microscope manufacturers 283,287,289,302,314,318,321,323, Bio-Rad filter wheel separation, 86, 363 Visitron Systems. See Confocal microscope Leica prism separation, 81, 403, 404, 405, 406 343 manufacturers Olympus diffiaction grating separation, 84,423 Airy disk, 115 Vital Images. See Imaging software Zeiss multi-PMT array, 84, 389, 390, 397, 401 in backscaltered light imaging, 5 Vitamin C. See Antifade reagents SpectraSharp software (Bio-Rad), 364 microscope objective, 56 Vitamin E (Trolux). See Antifade reagents Spectrum ofvisihle light, 187 opticallimit, 27, 29, 51, 52 VitroCom glass tubing, 288, 344 Spherieal aberration, 44, 48, 49, 58, 284, optimal zoom, 124, 125, 126, 127 VoxBlast. See Imaging software 315,317,318,350 ROB colour, 156 VoXEl View. See Imaging software Standard sampies. See Test samples Roc1cing motion image (3D representation), Wallac. See Confocal microscope Statistics (Poisson noise), 36, 37 137 manufacturers, PerkinElmer Stereo pair image, 22, 139 Scan head, 70, 71 Warner Instruments microscopy chambers, Streptavidin, 267 Scan rotation, 359, 404, 422, 425 305, 306, 333, 345 Streptolysin-O (SLO), 267, 277, 316 Sean speed, 116-20 Water immersion Objective lens. See Bio-Rad Radiance scan rates, 118 Surface label, 15, 18, 19,20,249,267,277 Objective lens decreased scan speed, 119 Sylgard silicone rubber, 285 Wave nature oflight, 32 fast scanning by decreasing collection box size, Teflon® film ehamber cover, 298,310,311, Wavelength oflight, 31, 187 140 313 fast scanning on an LSCM, 119, 120 Willco Wells microscopy dishes, 287, 302, Temperature high-speed Nipkow disk, 118, 140 344 Leica scan rates, 118 enzyme changes, 290 Temperature contro!. See Live cell and tissue Wollaston prism, 46, 109,390,407 optimal scan speed, 118 Xenon arc lamp, 63 slow-speed scanning, 119 imaging Yellow, in confocal microscopy, 128,247 Scanuing mirrors, 7, 8, 66, 71,117,359,366, Test sampies, 141-42, 141-42 Y okogawa. See Confocal mieroscope 385,404,417,422,425 fluorescent plastic, 141 manufacturers Scanuing zoom, 125 fluorescent solution, 141 hair autofluorescence. 142 Zeiss. See Confocal microscope Scion Image. See Imaging software latex beads (microspheres), 141 manufacturers, Carl Zeiss Sereen averaging, 121, 122, 263 moss spores, 142 Zoom Screen dots (dpi), 147, 148 pollen grains, 142 computer (digital) zoom, 127 Screen pixels, 149 your own cells, 142 confocal (scanning) zoom, 125-27, 134 Screen resolution display (1 to I), 134 Thirty-two-bit images (32-bit), 114 digital zoom, 127 Sea Pansy (Renilla reniformis) fluorescent Three dimensional (3D) image collection, for optimal resolution, 126 proteins, 233, 238 137 Semi-permeabilisation, 316 Three dimensional imaging, 4