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