DOCSLIB.ORG

Principles and Practices of Laser Scanning Confocal Microscopy Stephen W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Principles and Practices of Laser Scanning Confocal Microscopy Stephen W Laser Scanning Confocal MicroscopyREVIEW 127 Principles and Practices of Laser Scanning Confocal Microscopy Stephen W. Paddock* Abstract The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging appli- cations at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today’s sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstruc- tions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice. Index Entries: Confocal microscopy; laser scanning; fluorescence light microscopy. 1. Introduction field light microscope (theoretical maximum The major application of confocal microscopy resolution of 0.2 µm), but not as great as that in in the biomedical sciences is for imaging either the transmission electron microscope (0.1 nm), it fixed or living tissues that have been labeled with has bridged the gap between these two commonly one or more fluorescent probes. When these used techniques. samples are imaged using a conventional light The method of image formation in a confocal microscope, fluorescence in the specimen in focal microscope is fundamentally different from that planes away from the region of interest interferes in a conventional wide-field microscope where with resolution of structures in focus, especially the entire specimen is bathed in light from a mer- for those specimens that are thicker than 2 µm or cury or xenon source, and the image can be so (Fig. 1). The confocal approach provides a viewed directly by eye. In contrast, the illumina- slight increase in both lateral and axial resolution. tion in a confocal microscope is achieved by scan- It is the ability of the instrument to eliminate the ning one or more focused beams of light, usually “out-of-focus” flare from thick fluorescently from a laser, across the specimen. An image pro- labeled specimens that has caused the explosion duced by scanning the specimen in this way is in its popularity recently (1). Most modern con- called an optical section. This term refers to the focal microscopes are now relatively easy to noninvasive method of image collection by the operate and have become integral parts of instrument, which uses light rather than physical many multiuser imaging facilities. Since the means to section the specimen. The confocal resolution achieved by the LSCM is a little bet- approach has thus facilitated the imaging of liv- ter than that achieved in a conventional wide- ing specimens, enabled the automated collection *Author to whom all correspondence and reprint requests should be addressed. Department of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA, E-mail: [email protected] Molecular Biotechnology 2000 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2000/16:2/127–149/$15.75 MOLECULAR BIOTECHNOLOGY 127 Volume 16, 2000 128 Paddock Fig. 1. Conventional epifluorescence image (A) compared with a confocal image (B) of a similar region of a whole mount of a butterfly (Precis coenia) pupal wing stained with propidium iodide. Note the improved resolu- tion of the nuclear detail (B). of three-dimensional data in the form of Z-series, by Minsky, and all of the modern confocal imag- and improved the images of multiple labeled ing systems employ the principle of confocal specimens (1). imaging that he patented in 1957 (5). Emphasis has been placed on the laser scan- In Minsky’s original confocal microscope the ning confocal microscope (LSCM) throughout point source of light is produced by a pinhole this article because it is currently the instrument placed in front of a zirconium arc source. The of choice for most biomedical research applica- point of light is focused by an objective lens into tions, and it is therefore most likely to be the the specimen, and light that passes through it is instrument first encountered by the novice user. focused by a second objective lens at a second Several alternative designs of confocal instru- pinhole, which has the same focus as the first pin- ments occupy specific niches within the biologi- hole, i.e., it is confocal with it. Any light that cal imaging field (2). Optical sections can be passes through the second pinhole strikes a low- produced using other, nonconfocal, methods. For noise photomultiplier, which produces a signal example, using deconvolution, which calculates that is directly proportional to the brightness of the out-of-focus information in an image and the light. The second pinhole prevents light from removes it digitally (3), and multiple photon above or below the plane of focus from striking imaging (4), which uses the same method of scan- the photomultiplier. This is the key to the confo- ning as the LSCM but uses a laser that only cal approach, namely the elimination of out-of- excites the fluorochromes that are imaged in the focus light or “flare” in the specimen by spatial optical section itself. filtering. Minsky also described a reflected light version of the microscope that uses a single objec- 2. Evolution of the Confocal Approach tive lens and a dichromatic mirror arrangement. The development of confocal microscopes This is the basic configuration of most modern was driven largely by a desire to image biologi- confocal systems used for fluorescence imaging cal events as they occur in vivo. The invention (Fig. 2). of the confocal microscope is usually attributed In order to build an image, the focused spot of to Marvin Minsky, who built a working micro- light must be scanned across the specimen in scope in 1955 with the goal of imaging neural some way. In Minsky’s original microscope the networks in unstained preparations of living beam was stationary and the specimen itself was brains. Details of the microscope, and of its devel- moved on a vibrating stage. This optical arrange- opment can be found in an informative memoir ment has the advantage of always scanning on the MOLECULAR BIOTECHNOLOGY Volume 16, 2000 Laser Scanning Confocal Microscopy 129 image quality in his microscope was not very impressive because of the quality of the oscillo- scope display and not because of lack of resolu- tion achieved with the microscope itself. It is clear that the technology was not available to Minsky in 1955 to fully demonstrate the poten- tial of the confocal approach especially for imag- ing biological structures. According to Minsky, this is perhaps a reason why confocal microscopy did not immediately catch on with the biological community, who were, (as they are now) a highly demanding and fickle group when it came to the quality of their images. After all, at the time they could quite easily view and photograph their brightly stained and colorful histological tissue sec- tions using light microscopes with excellent optics. In modern confocal microscopes the image is either built up from the output of a photo- multiplier tube or captured using a digital charge coupled device (CCD) camera, directly pro- cessed in a computer imaging system, and then displayed on a high-resolution video monitor and recorded on modern hard copy devices, with Fig. 2. The light path in a typical LSCM is based upon that of a conventional reflected-light wide-field spectacular results. epifluorescence microscope, but with pinholes placed The optics of the light microscope have not in front of the light source (the point source is now a changed drastically in decades since the final laser) and in front of the photodetector. The pinhole at resolution achieved by the instrument is governed the light source, the focused point in the specimen, by the wavelength of light, the objective lens and and the pinhole in front of the detector are all confocal properties of the specimen itself. However, the with one another. associated technology and the dyes used to add contrast to the specimens have been improved sig- optical axis, which can eliminate any lens defects. nificantly over the past 20 yr. The confocal However, for many biological specimens, move- approach is a direct result of a renaissance in ment of the specimen can cause them to wobble light microscopy that has been fueled largely by and distort, which results in a loss of resolution in advancements in modern technology. Several the image. Moreover, it is impossible to perform major technological advances that would have various manipulations such as microinjection of benefited Minsky’s confocal design are now fluorescently labeled probes when the specimen available (and affordable) to biologists. These is moving. advances include: Finally an image of the specimen has to be pro- 1. Stable multiwavelength lasers for brighter duced. A real image is not formed in Minsky’s point sources of light. original microscope but rather the output from 2. More efficiently reflecting mirrors. the photodetector is translated into an image of 3. Sensitive low-noise photodetectors. the region-of-interest. In Minsky’s original design 4. Fast microcomputers with image processing the image was built up on the screen of a military capabilities. surplus oscilloscope with no facility for hard 5. Elegant software solutions for analyzing the copy. Minsky admitted at a later date that the images. MOLECULAR BIOTECHNOLOGY Volume 16, 2000 130 Paddock 6. High-resolution video displays and digital of confocal instrumentation have been covered printers. elsewhere (2), but briefly there are two funda- 7. Brighter and more stable fluorescent probes. mentally different methods of beam scanning: multiple-beam scanning or single-beam scan- These technologies have been developed inde- ning. The most popular way is currently single- pendently, and since 1955, they have slowly been beam scanning, which is typified by the LSCM.
Load more

Recommended publications
  • Second Harmonic Imaging Microscopy
    170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison.
    [Show full text]
  • Multiphoton Microscopy
    Living up to Life Multiphoton Microscopy 1 Jablonski Diagram: Living up to Life Nonlinear Optical Microscopy F.- Helmchen, W. Denk, Deep tissue two-photon microscopy, Nat. Methods 2, 932-940 2 Typical Samples – Living up to Life Small Dimensions & Highly Scattering • Somata 10-30 µm • Dendrites 1-5 µm • Spines ~0.5 µm • Axons 1-2 µm ls ~ 50-100 µm (@ 630 nm) ls ~ 200 µm (@ 800 nm) T. Nevian Institute of Physiology University of Bern, Switzerland F.- Helmchen, W. Denk Deep tissue two-photon microscopy. Nat. Methods 2, 932-940 3 Why Multiphoton microscopy? Living up to Life • Today main challenge: To go deeper into samples for improved studies of cells, organs or tissues, live animals Less photodamage, i.e. less bleaching and phototoxicity • Why is it possible? Due to the reduced absorption and scattering of the excitation light 4 The depth limit Living up to Life • Achievable depth: ~ 300 – 600 µm • Maximum imaging depth depends on: – Available laser power – Scattering mean-free-path – Tissue properties • Density properties • Microvasculature organization • Cell-body arrangement • Collagen / myelin content – Specimen age – Collection efficiency Acute mouse brain sections containing YFP neurons,maximum projection, Z stack: 233 m Courtesy: Dr Feng Zhang, Deisseroth laboratory, Stanford University, USA Page 5 What is Two‐Photon Microscopy? Living up to Life A 3-dimensional imaging technique in which 2 photons are used to excite fluorescence emission exciting photon emitted photon S1 Simultaneous absorption of 2 longer wavelength photons to
    [Show full text]
  • Adaptive Optics in Microscopy
    Downloaded from http://rsta.royalsocietypublishing.org/ on February 3, 2015 Phil. Trans. R. Soc. A (2007) 365, 2829–2843 doi:10.1098/rsta.2007.0013 Published online 13 September 2007 Adaptive optics in microscopy BY MARTIN J. BOOTH* Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK The imaging properties of optical microscopes are often compromised by aberrations that reduce image resolution and contrast. Adaptive optics technology has been employed in various systems to correct these aberrations and restore performance. This has required various departures from the traditional adaptive optics schemes that are used in astronomy. This review discusses the sources of aberrations, their effects and their correction with adaptive optics, particularly in confocal and two-photon microscopes. Different methods of wavefront sensing, indirect aberration measurement and aberration correction devices are discussed. Applications of adaptive optics in the related areas of optical data storage, optical tweezers and micro/nanofabrication are also reviewed. Keywords: adaptive optics; aberrations; confocal microscopy; multiphoton microscopy; optical data storage; optical tweezers 1. Introduction Optical microscopes are essential tools in many scientific fields. In the life sciences, they are widely used for the visualization of cellular structures and sub- cellular processes. Confocal and multiphoton microscopes are particularly important in this respect as they produce three-dimensional images of volumetric objects. However, the resolution of these microscopes is often adversely affected by the optical properties of the specimen itself. Spatial variations in the refractive index of the specimen introduce optical aberrations that compromise image quality. This is a particular problem when imaging deep into thick biological specimens.
    [Show full text]
  • Fv1000 Fluoview
    Confocal Laser Scanning Biological Microscope FV1000 FLUOVIEW FLUOVIEW—Always Evolving FLUOVIEW–—From Olympus is Open FLUOVIEW—More Advanced than Ever The Olympus FLUOVIEW FV1000 confocal laser scanning microscope delivers efficient and reliable performance together with the high resolution required for multi-dimensional observation of cell and tissue morphology, and precise molecular localization. The FV1000 incorporates the industry’s first dedicated laser light stimulation scanner to achieve simultaneous targeted laser stimulation and imaging for real-time visualization of rapid cell responses. The FV1000 also measures diffusion coefficients of intracellular molecules, quantifying molecular kinetics. Quite simply, the FLUOVIEW FV1000 represents a new plateau, bringing “imaging to analysis.” Olympus continues to drive forward the development of FLUOVIEW microscopes, using input from researchers to meet their evolving demands and bringing “imaging to analysis.” Quality Performance with Innovative Design FV10i 1 Imaging to Analysis ing up New Worlds From Imaging to Analysis FV1000 Advanced Deeper Imaging with High Resolution FV1000MPE 2 Advanced FLUOVIEW Systems Enhance the Power of Your Research Superb Optical Systems Set the Standard for Accuracy and Sensitivity. Two types of detectors deliver enhanced accuracy and sensitivity, and are paired with a new objective with low chromatic aberration, to deliver even better precision for colocalization analysis. These optical advances boost the overall system capabilities and raise performance to a new level. Imaging, Stimulation and Measurement— Advanced Analytical Methods for Quantification. Now equipped to measure the diffusion coefficients of intracellular molecules, for quantification of the dynamic interactions of molecules inside live cell. FLUOVIEW opens up new worlds of measurement. Evolving Systems Meet the Demands of Your Application.
    [Show full text]
  • Imaging with Second-Harmonic Generation Nanoparticles
    1 Imaging with Second-Harmonic Generation Nanoparticles Thesis by Chia-Lung Hsieh In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2011 (Defended March 16, 2011) ii © 2011 Chia-Lung Hsieh All Rights Reserved iii Publications contained within this thesis: 1. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging," Opt. Express 17, 2880–2891 (2009). 2. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes," Biomaterials 31, 2272–2277 (2010). 3. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Second harmonic generation from nanocrystals under linearly and circularly polarized excitations," Opt. Express 18, 11917–11932 (2010). 4. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media," Opt. Express 18, 12283–12290 (2010). 5. C. L. Hsieh, Y. Pu, R. Grange, G. Laporte, and D. Psaltis, "Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle," Opt. Express 18, 20723–20731 (2010). iv Acknowledgements During my five-year Ph.D. studies, I have thought a lot about science and life, but I have never thought of the moment of writing the acknowledgements of my thesis. At this moment, after finishing writing six chapters of my thesis, I realize the acknowledgment is probably one of the most difficult parts for me to complete.
    [Show full text]
  • Contrast Enhancement for Topographic Imaging in Confocal Laser Scanning Microscopy
    applied sciences Article Contrast Enhancement for Topographic Imaging in Confocal Laser Scanning Microscopy Lena Schnitzler *,†, Markus Finkeldey †, Martin R. Hofmann and Nils C. Gerhardt Photonics and Terahertz Technology, Ruhr University Bochum, 44780 Bochum, Germany * Correspondence: [email protected] † These authors contributed equally to this work. Received: 26 June 2019; Accepted: 29 July 2019; Published: 31 July 2019 Featured Application: The method for contrast enhancement presented in this work, can be used to increase the image contrast of topographic structures. It might be an essential tool for non-destructive testing in the quality management of microcontroller production, in the processing of semiconductors or in the developement and characterization of MEMs. Even structures with low height variance can be observed. Abstract: The influence of the axial pinhole position in a confocal microscope in terms of the contrast of the image is analyzed. The pinhole displacement method is introduced which allows to increase the contrast for topographic imaging. To demonstrate this approach, the simulated data of a confocal setup as well as experimental data is shown. The simulated data is verified experimentally by a custom stage scanning reflective microscopy setup using a semiconductor test target with low contrast structures of sizes between 200 nm and 500 nm. With the introduced technique, we are able to achieve a contrast enhancement of up to 80% without loosing diffraction limited resolution. We do not add additional components to the setup, thus our concept is applicable for all types of confocal microscopes. Furthermore, we show the application of the contrast enhancement in imaging integrated circuits. Keywords: microscopy; confocal microscopy; contrast enhancement 1.
    [Show full text]
  • Optical Tweezers and Confocal Microscopy for Simultaneous Three-Dimensional Manipulation and Imaging in Concentrated Colloidal Dispersions Dirk L
    REVIEW OF SCIENTIFIC INSTRUMENTS VOLUME 75, NUMBER 9 SEPTEMBER 2004 Optical tweezers and confocal microscopy for simultaneous three-dimensional manipulation and imaging in concentrated colloidal dispersions Dirk L. J. Vossena) and Astrid van der Horst FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, and Soft Condensed Matter, Debye Institute, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands Marileen Dogterom FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands Alfons van Blaaderenb) FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, and Soft Condensed Matter, Debye Institute, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands (Received 25 February 2004; accepted 10 June 2004; published 14 September 2004) A setup is described for simultaneous three-dimensional manipulation and imaging inside a concentrated colloidal dispersion using (time-shared) optical tweezers and confocal microscopy. The use of two microscope objectives, one above and one below the sample, enables imaging to be completely decoupled from trapping. The instrument can be used in different trapping (inverted, upright, and counterpropagating) and imaging modes. Optical tweezers arrays, dynamically changeable and capable of trapping several hundreds of micrometer-sized particles, were created using acousto-optic deflectors. Several schemes are demonstrated to trap three-dimensional colloidal structures with optical tweezers. One combined a Pockels cell and polarizing beam splitters to create two trapping planes at different depths in the sample, in which the optical traps could be manipulated independently. Optical tweezers were used to manipulate collections of particles inside concentrated colloidal dispersions, allowing control over colloidal crystallization and melting.
    [Show full text]
  • Introduction to Confocal Laser Scanning Microscopy (LEICA)
    Introduction to Confocal Laser Scanning Microscopy (LEICA) This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics. For questions & registration please contact www.zmb.unizh.ch . Confocal Laser Scanning Microscopy xy yz 100 µm xz 100 µm xy yz xz thick specimens at different depth 3D reconstruction Types of confocal microscopes { { { point confocal slit confocal spinning disc confocal (Nipkov) Best resolution and out-of-focus suppression as well as highest multispectral flexibility is achieved only by the classical single point confocal system ! Fundamental Set-up of Fluorescence Microscopes: confocal vs. widefield Confocal Widefield Fluorescence Fluorescence Microscopy Microscopy Photomultiplier LASER detector Detector pinhole aperture CCD Dichroic mirror Fluorescence Light Source Light source Okular pinhole aperture Fluorescence Filter Cube Objectives Sample Plane Z Focus Confocal laser scanning microscope - set up: The system is composed of a a regular florescence microscope and the confocal part, including scan head, laser optics, computer. Comparison: Widefield - Confocal Y X Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer. Only a small volume can be visualized by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling.
    [Show full text]
  • Label-Free Multiphoton Microscopy: Much More Than Fancy Images
    International Journal of Molecular Sciences Review Label-Free Multiphoton Microscopy: Much More than Fancy Images Giulia Borile 1,2,*,†, Deborah Sandrin 2,3,†, Andrea Filippi 2, Kurt I. Anderson 4 and Filippo Romanato 1,2,3 1 Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy; fi[email protected] 2 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy; [email protected] (D.S.); andrea.fi[email protected] (A.F.) 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, 35128 Padua, Italy 4 Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London NW1 1AT, UK; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment.
    [Show full text]
  • Combined Two-Photon Excited Fluorescence and Second-Harmonic Generation Backscattering Microscopy of Turbid Tissues
    UC Irvine ICTS Publications Title Combined two-photon excited fluorescence and second-harmonic generation backscattering microscopy of turbid tissues Permalink https://escholarship.org/uc/item/2q9857fp Journal Proceedings of SPIE - The International Society for Optical Engineering, 4620 Authors Zoumi, A Yeh, AT Tromberg, BJ Publication Date 2002 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Combined Two-Photon Excited Fluorescence and Second-Harmonic Generation Backscattering Microscopy of Turbid Tissues Aikaterini Zoumi a, b, Alvin T. Yeh a, and Bruce J. Tromberg a, b * aLaser Microbeam and Medical Program (LAMMP), Beckman Laser Institute, University of California, Irvine, CA 92612. bCenter for Biomedical Engineering, University of California, Irvine, CA 92612. ABSTRACT A broad range of excitation wavelengths (730-880nm) was used to demonstrate the co-registration of two- photon excited fluorescence (TPEF) and second-harmonic generation (SHG) in unstained turbid tissues in reflection geometry. The composite TPEF/SHG microscopic technique was applied to imaging an organotypic tissue model (RAFT). The origin of the image-forming signal from the various RAFT constituents was determined by spectral measurements. It was shown that at shorter excitation wavelengths the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. The cellular signal is due to TPEF at all excitation wavelengths. The reflected SHG intensity followed a quadratic dependence on the excitation power and exhibited a spectral dependence in accordance with previous theoretical studies.
    [Show full text]
  • Lasers for Confocal Microscopy All Colors of the Rainbow © Anja Kaiser – Fotolia.Com ©
    Lasers for Confocal Microscopy All Colors of the Rainbow © Anja Kaiser – Fotolia.com © Easy-to-use new lasers are more and more replacing bulky legacy laser systems. This new generation of Keywords lasers provides flexibility, easy operation and is all in all more convenient to use. These novel approaches Laser, Microscopy, Fluorescence, Multicolor offer also new possibilities – supporting the rapid development of new microscopy techniques. Here we will review different laser types and their fields of application. scanning confocal microscope used an ser diodes to directly produce the output air-cooled argon laser for excitation. In wavelength; however for years, it was im- comparison with other light sources la- possible to directly generate wavelengths sers provide a high brightness, a small di- in the blue and green spectral range. In vergence and they are highly monochro- 1998 the emergence of the semiconduc- matic, properties which make them ideal tor material Gallium Nitride (originally light sources for confocal microscopy. developed for the optical data storage in- Gas Lasers such as Ar-ion, Kr-ion and dustry) facilitated the direct generation others are still widely used for micros- of blue wavelengths. These new diode la- Dr. Marion Lang, copy as for a long time only these were sers, above all 488 nm, were quickly es- Technical Marketing Manager, Toptica able to provide the colors and beam tablished. Figure 1 shows a confocal im- Photonics, Gräfelfing, Germany quality required. Occasionally dye lasers age of rat brain axons acquired with the are employed which consist of a solution iBeam smart 488 nm diode laser.
    [Show full text]
  • Sensor Characterization by Comparative Measurements Using a Multi-Sensor Measuring System
    J. Sens. Sens. Syst., 8, 111–121, 2019 https://doi.org/10.5194/jsss-8-111-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Sensor characterization by comparative measurements using a multi-sensor measuring system Sebastian Hagemeier, Markus Schake, and Peter Lehmann Measurement Technology Group, University of Kassel, 34121 Kassel, Germany Correspondence: Sebastian Hagemeier ([email protected]) Received: 7 September 2018 – Accepted: 8 February 2019 – Published: 28 February 2019 Abstract. Typical 3-D topography sensors for the measurement of surface structures in the micro- and nanome- tre range are atomic force microscopes (AFMs), tactile stylus instruments, confocal microscopes and white-light interferometers. Each sensor shows its own transfer behaviour. In order to investigate transfer characteristics as well as systematic measurement effects, a multi-sensor measuring system is presented. With this measurement system comparative measurements using five different topography sensors are performed under identical con- ditions in a single set-up. In addition to the concept of the multi-sensor measuring system and an overview of the sensors used, surface profiles obtained from a fine chirp calibration standard are presented to show the diffi- culties of an exact reconstruction of the surface structure as well as the necessity of comparative measurements conducted with different topography sensors. Furthermore, the suitability of the AFM as reference sensor for high-precision measurements is shown by measuring the surface structure of a blank Blu-ray disc. 1 Introduction are eliminated. For the compensation of disturbances caused by external vibrations, different methods exist (Tereschenko The characterization of surface structures in the micro- and et al., 2016; Seewig et al., 2013; Kiselev et al., 2017; Schake nanometre range can be done by various types of topogra- and Lehmann, 2018).
    [Show full text]