confocal microscopy

Preface This Methods in Enzymology volume deals with the rapidly evolving topic of confocal microscopy. The OVID database (including MEDLINE, Current Contents, and other sources) lists 76 references to confocal micros- copy for the five-year period 1985-1989. In contrast, for the four-year period 1995-1998, nearly 3600 references are listed. This volume documents many diverse uses for confocal microscopy in disciplines that broadly span biology. The methods presented include shortcuts and conveniences not included in the sources from which they were taken. The techniques are described in a context that allows compari- sons to other related methodologies. The authors were encouraged to do this in the belief that such comparisons are valuable to readers who must adapt extant procedures to new systems. Also, so far as possible, methodolo- gies are presented in a manner that stresses their general applicability and potential limitations. Although for various reasons some topics are not covered, the volume provides a substantial and current overview of the extant methodology in the field and a view of its rapid development. Particular thanks go to the authors for their attention to meeting dead- lines and for maintaining high standards of quality, to the series editors for their encouragement, and to the staff of Academic Press for their help and timely publication of the volume. P. MICHAEL CONN xiii Contributors to Volume 307 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. JOHN H. ANDREWS (34), Department of Plant Pathology, University of Wisconsin, Madi- son, Wisconsin 53706 SILVIA M. ARRIBAS (15), Departamento de Fisiologia, Facultad de Medicina, Universi- dad Aut6noma de Madrid, 28029 Madrid, Spain GEORGE F. BABCOCK (18), Departments of Surgery and Cell Biology, University of Cin- cinnati College of Medicine, and Shriners Hospitals for Children, Cincinnati Burns In- stitute, Cincinnati, Ohio 45267-0558 WERNER BASCHONG (11), M. E. Miiller Insti- tute for Structural Biology and Department of Oral Surgery, Biozentrum of the Univer- sity of Basel, CH-4056 Basel, Switzerland MIGUEL BERRIOS (4), Department of Pharma- cological Sciences and University Micros- copy Imaging Center, University Hospital and Medical Center, State University of New York, Stony Brook, New York 11794-8088 KANTI D. BHOOLA (22), Department of Exper- imental and Clinical Pharmacology, Faculty of Medicine, University of Natal, Congella 4001, South Africa MIKE BIRCH (28), Unit of Ophthalmology, Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom GHASSAN BKAILY (8), MRCC Group in Ira- roUnD-Cardiovascular Interactions, Depart- ment of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QuEbec, Canada JIH 5N4 LOTHAR A. BLATTER (16), Department of Physiology, Loyola University of Chicago, Maywood, Illinois 60153 MATIqqIAS BOHNKE (30), Department of Ophthalmology, University of Bern, 3010 Bern, Switzerland ix ALBERICO BORGHETrI (20), Department of Clinical Medicine, Nephrology, and Health Sciences, University of Parma, 43100 Parma, Italy DAVID N. BOWSER (25), Confocal and Fluo- rescence Imaging Group, Department of Physiology, The University of Melbourne, Parkville, Victoria 3052, Australia DANIEL BROTCHIE (28), Unit of Ophthalmol- ogy, Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom CHRISTOF BUEHLER (29), Department of Me- chanical Engineering, Massachusetts Insti- tute of Technology, Cambridge, Massachu- setts 02139 NICK CALLAMARAS (10), Department of Neu- robiology and Behavior, University of Cali- fornia, Irvine, California 92697-4550 SILVANO CAPITANI (12), Institute of Human Anatomy, University of Ferrara, 44100 Fer- rata, Italy H. DWIGHT CAVANAGH (14), Department of Ophthalmology, University of Texas South- western Medical Center, Dallas, Texas 75235-9057 CATERINA CINTI (12), Institute of Citomorfo- logia Normale e Patologica, C.N.R., 66100 Chieti, Italy DAVID E. COLFLESH (4), University Micros- copy Imaging Center, University Hospital and Medical Center, State University of New York, Stony Brook, New York 11794-8088 KIMBERLY A. CONLON (4), Department of Pharmacological Sciences, University Hos- pital and Medical Center, State University of New York, Stony Brook, New York 11794-8651 GuY Cox (3), Electron Microscope Unit, Uni- versity of Sydney, Sydney, New South Wales 2006, Australia X CONTRIBUTORS TO VOLUME 307 DANIEL CULLEN (34), Forest Products Labo- ratory, U.S. Department of Agriculture For- est Service, Madison, Wisconsin 53705 CRAIG J. DALY (15), Autonomic Physiology Unit, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom MARKUS DI~RRENBERGER (11), Interdivi- sional Electron Microscopy, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland RALE ENGELMANN (31), Leibniz-Institut for Neurobiology, D-39118 Magdeburg, Ger- many MARGHERITA FONTANA (27), Oral Biology and Oral Health Research Institute, Indiana University School of Dentistry, Indianapo- lis, Indiana 46202 RITA GATrI (20), Institute of Histology and General Embryology, University of Parma, 43100 Parma, Italy GIAN CARLO GAZZOLA (20), Institute of Gen- eral Pathology, University of Parma, 43100 Parma, Italy CARLOS GONZ,~EZ-CABEZAS (27), Oral Biol- ogy and Oral Health Research Institute, In- diana University School of Dentistry, India- napolis, Indiana 46202 ENRICO GRATTON (29), Laboratory for Flu- orescence Dynamics, Department of Physics, University of Illinois at Urbana- Champaign, Urbana, Illinois 61801 IAN GRIERSON (28), Unit of Ophthalmology, Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom HISASHI HASHIMOTO (6), Department of Anat- omy, The Jikei University School of Medi- cine, Minato-ku, Tokyo 105-8461, Japan, and Center for Biogenic Resources, The In- stitute of Physical and Chemical Research (RIKEN), Tsukuba, Ibarald 305-0074, Japan PENNY HOGG (28), Unit of Ophthalmology, Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom C. VYVYAN HOWARD (28), Department of Fe- tal and Infant Toxico-Pathology, University of Liverpool, Liverpool L69 3GA, United Kingdom DAVID N. HOWELL (32), Departments of Pa- thology and Laboratory Medicine Service, Veterans Affairs Medical Center, Durham, North Carolina 27705, and Duke University Medical Center, Durham, North Carolina 27710 JoN R. INGLEEIELD (26), Neurotoxicology Di- vision, National Health and Environmental Effects Research Laboratory, U.S. Environ- mental Protection Agency, Research Trian- gle Park, North Carolina 27711 HIROSHI ISHIKAWA (6), Department of Anat- omy, The Jikei University School of Medi- cine, Minato-ku, Tokyo 105-8461, Japan DANIELLE JACQUES (8), MRCC Group in Ira- rounD-Cardiovascular Interactions, Depart- ment of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4 JAMES V. JESTER (14), Department of Oph- thalmology, University of Texas Southwest- ern Medical Center, Dallas, Texas 75235- 9057 MANABU KAGAYAMA (5), Department of Anatomy, Tohoku University School of Dentistry, Sendai 980-8575, Japan KI HEAN KIM (29), Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 SUSAN M. KNOBEL (21), Department of Mo- lecular Physiology and Biophysics, Vander- bilt University, Nashville, Tennessee 37232 SAMUEL KO (2), Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong S. K. KONG (2), Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong ULRICH KUBITSCHECK (13), Institut far Medi- zinische Physik und Biophysik, Universiti~t Mtinster, D-48149 Mtinster, Germany CONTRIBUTORS TO VOLUME 307 xi THORSTEN KUES (13), Institut far Medizin- ische Physik und Biophysik, Universitat Miinster, D-48149 Miinster, Germany MORIAKI KUSAKABE (6), Center for Biogenic Resources, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305-0074, Japan C. Y. LEE (2), Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong P. Y. Lui (2), Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong ANNA MANDINOVA (11), M. E. Miiller Insti- tute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland CARLOS B. MANTILLA (17), Mayo Clinic, Rochester, Minnesota 55905 FRANCESCO A. MANZOLI (12), Institute of Human Anatomy, University of Bologna, 40126 Bologna, Italy NADIR M. MARALDI (12), Institute of Cito- morfologia Normale e Patologica, C.N.R., and Laboratory of Biologia Cellulare e Microscopia Elettronica, L O.R., 40136 Bologna, Italy, and Institute of Human Anatomy, University of Bologna, 40123 Bologna, Italy BARRY R. MASTERS (29, 30), Department of Mechanical Engineering, Massachusetts In- stitute of Technology, Cambridge, Massa- chusetts 02139, and Department of Ophthal- mology, University of Bern, 3010 Bern, Switzerland JOHN C. MCGRA77-I (15), Autonomic Physiol- ogy Unit, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom SARA E. MILLER (32), Departments of Micro- biology and Pathology, Duke University Medical Center, Durham, North Carolina 27710 KAZUTAKA MOMOSE (24), Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan LUCA M. NERI (12), Institute of Human Anatomy, University of Ferrara, 44100 Fer- rara, Italy HISAVUKI OHATA (24), Department of Phar- macology, School of Pharmaceutical Sci- ences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan GUIDO ORLANDINI (20), Department of Clini- cal Medicine, Nephrology, and Health Sci- ences, University of Parma, 43100 Parma, Italy IAN PARKER (10), Department of Neurobiol- ogy and Behavior, University of California, Irvine, California 92697-4550 REINER PETERS (13), Institutfar Medizinische Physik und Biophysik, Universiti~t Miinster, D-48149 MUnster, Germany W. MATI'HEW PETROLL (14), Department of Ophthalmology, University of Texas South- western Medical Center, Dallas, Texas 75235-9057 STEVEN PETROU (25), Confocal and Fluores- cence Imaging Group, Department of Phys- iology, The University of Melbourne, Park- viUe, Victoria 3052, Australia DAVID W. PISTON (21), Department of Molec- ular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 TORSTEN PORWOL (7), Max-Planck-Institut far Molekulare Physiologie, D-44202 Dort- mund, Germany PIERRE POTHIER (8), MRCC Group in Ira- rounD-Cardiovascular Interactions, Depart- ment of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QuEbec, Canada J1H 5N4 Y. S. PRAKASH (17), Mayo Clinic, Rochester, Minnesota 55905 DESHANDRA M. RAIDOO (22), Department of Experimental and Clinical Pharmacology, Faculty of Medicine, University of Natal, Congella 4001, South Africa GOUSEI RIE (24), Department of Pharmacol- ogy, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan xii CONTRIBUTORS TO VOLUME 307 NElL ROBERTS (28), Magnetic Resonance Re- search Centre, University of Liverpool, Liv- erpool L69 3GA, United Kingdom NICOLETTA RONDA (20), Department of Clini- cal Medicine, Nephrology, and Health Sci- ences, University of Parma, 43100 Parma, Italy BERNHARD A. SABLE (31), Institute of Medi- cal Psychology, Otto-v Guericke Univer- sity of Magdeburg, D-39120 Magdeburg, Germany SPARTACO SANTI (12), Institute of Citomorfo- logia Normale e Patologica, C.N.R., 66100 Chieti, Italy YASUYUKI SASANO (5), Department of Anat- omy, Tohoku University School of Den- tistry, Sendai 980-8575, Japan ROCHELLE D. SCHWARTZ-BLOOM (26), De- partment of Pharmacology and Cancer Bi- ology, Duke University Medical Center, Durham, North Carolina 27710 AKIHISA SEGAWA (19), Department of Anat- omy, School of Medicine, Kitasato Uni- versity, Sagamihara, Kanagawa 228-8555, Japan GARY C. SIECK (17), Mayo Clinic, Rochester, Minnesota 55905 GEOFFREY L. SMITH (33), Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom CELIA J. SNYMAN (22), Department of Experi- mental and Clinical Pharmacology, Faculty of Medicine, University of Natal, Congella 4001, South Africa PETER T. C. So (29), Department of Mechani- cal Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 RUSSELL N. SPEAR (34), Department of Plant Pathology, University of Wisconsin, Madi- son, Wisconsin 53706 EBERHARD SPIESS (7), Biomedizinische Strukturforschung, Deutsches Krebs- forschungszentrum, D-69009 Heidelberg, Germany STEFANO SQUARZONI (12), Institute of Cito- morfologia Normale e Patologica, C.N.R., 40136 Bologna, Italy GEORGE K. STOOKEY (27), Oral Biology and Oral Health Research Institute, Indiana University School of Dentistry, Indianapo- lis, Indiana 46202 ANJA-ROSE STROHMAIER (7), Nikon GmbH, D-40472 Dasseldorf Germany LIBORIO STUPPIA (12), Institute of Biologia e Genetica, University "G. d'Annunzio" 66100 Chieti, Italy ROSMARIE SUETFERLIN (11), M. E. Mallet In- stitute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Ba- sel, Switzerland XUEJUN SUN (9), Department of Oncology, University of Alberta, Cross Cancer In- stitute, Edmonton, Alberta T6G 1Z2, Can- ada YOSUKE UJIKE (24), Department of Pharma- cology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan ALAIN VANDERPLASSCHEN (33), Immunol- ogy-Vaccinology, Faculty of Veterinary Medicine, University of Liege, B-4000 LiOge, Belgium ROBERT H. WEBB (1), Schepens Eye Research Institute, and Wellman Laboratories of Pho- tomedicine, Massachusetts General Hospi- tal, Boston, Massachusetts 02114 DAVID ALAN WILLIAMS (25), Confocal and Fluorescence Imaging Group, Depart- ment of Physiology, The University of Melbourne, Parkville, Victoria 3052, Aus- tralia KAZUHIRO YAMAGUCHI (23), Department of Medicine, School of Medicine, Keio Univer- sity, Tokyo 160-8582, Japan MASAYUKI YAMAMOTO (24), Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan [ 11 THEORETICAL BASIS 3 [1] Theoretical Basis of Confocal Microscopy By ROBERT H. WEBB A Simple View A confocal microscope is most valuable in seeing clear images inside thick samples. To demonstrate this, I want to start with a conventional wide-field epifluorescence microscope shown in Fig. 1. The left diagram (Fig. 1) demonstrates the illumination light, and the right shows light col- lected from the sample. In the right diagram we see that a broad field of illumination is imaged into the thick sample. Although the illumination is focused at one plane of the sample, it lights up all of the sample. In the right diagram we see that the microscope objective has formed the image of the whole thick sample at the image plane of the microscope. If we put a film, charge-coupled device (CCD), or retina at the image plane, it will record the in-focus image of one plane within the thick sample, but it will also record all of those out-of-focus images of the other planes. In Fig. 2 I show an alternative arrangement. Instead of a broad light source, I use a single point source of light and image it inside the thick sample. That focused light illuminates a single point inside the sample very brightly, but of course it also illuminates the rest of the sample at least weakly. On the right (Fig. 2) the image of the thick sample is very bright where the sample was brightly illuminated and dimmer where it was weakly illuminated. Since my intention is to look only at one point inside the thick sample, I will now put a pinhole in the image plane. The pinhole lets through only the light that is forming the bright part of the image. Behind the pinhole I put a detector, as shown in Fig. 3. That detector registers the brightness of the part of the thick sample that is illuminated by the focused light and ignores the rest of the sample. What we have here is a point source of light, a point focus of light inside the object or sample, and a pinhole detector, all three confocal with each other. That is a confocal microscope.l-3 This confocal microscope has all the features we need for looking at a point inside a thick sample. However, it is not very interesting to look at a single point. So we have to find a way to map out the whole sample point 1 j. Pawley, ed., in "Handbook of Biological Confocal Microscopy," 3rd ed. Plenum, New York 1996. 2 T. Wilson, ed., in "Confocal Microscopy." Academic Press, London, 1990. 3 R. H. Webb, Rep. Prog. Phys. 59, 427 1996. Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 307 0076-6879/99 $30.00 4 THEORY AND PRACTICAL CONSIDERATIONS [ 1] Image of thick sample . " Extended light source f, Thick sample o . - . o o * . -o . Illumination light path Collection light path Fro. 1. A conventional (wide-field) microscope for fluorescence in epitaxial configuration. by point. Most laser-scanning confocal microscopes look at one point of the sample at a time. Other varieties look at many well-separated points at once, but locally they are imaging one point at a time. The easiest way to look around in the sample is to move the sample, a technique called stage scanning. More complex scanning means allow the sample to be stationary while we move the illuminated spot(s) over the sample. But those are engineering details. Instead of concerning ourselves with them at the moment, let us assume that they are solved and investigate what properties this confocal microscope has. Optical Sectioning Our microscope discriminates against points near, but not in, the focal spot. When the unwanted points are beside the focal spot, the contrast has improved. However, this device also discriminates against points above and below the focus, a feature we call optical sectioning. Instead of using a microtome to slice a thin section out of a thick sample, we can now image that thin section inside the sample. Parts of the sample that are above the [ 11 THEORETICAL BASIS 5 Image of illuminated sample Point light source . _ -_ _ " - ° . - . .'-" ° .°_. - ° ." . " ? . ° . Collection light path F1G. 2. The microscope of Fig. 1 with point illumination. imaged point or below it will be illuminated weakly, and light from those parts will be mostly rejected by the pinhole. With scanning, this microscope can image a whole plane inside a thick sample and then be focused deeper into the sample to image a different layer, and those two images do not interfere with each other. With proper controls, the microscope can image a whole stack of optical sections, which can later be assembled into a three- dimensional display. 4'5 Figure 4 shows an even more abstract sketch of a confocal microscope that emphasizes optical sectioning. An object in the sample that lies above the focal point is imaged above the pinhole. Light going toward that image is mostly blocked by the pinhole mask. The confocal microscope also rejects light from points adjacent to the one illuminated. That increases the contrast, even for thin samples. Contrast enhancement is always desirable, particularly when we need to look at something dim next to something bright. This fact explains why confocal 4 G. J. Brakenhoff et al., Scann. Microsc. 2, 1831 (1988). 5 F. E. Morgan et al., Scann. Microsc. 6, 345 (1992). Image of illuminated sample point is all that gets through the pinhole ~.~ I Detector (PMT) [ ght source \ ~ Brightly illuminated .~point in sample Illumination light path 6 THEORY AND PRACTICAL CONSIDERATIONS [ 11 Collection light path FIG. 3. The microscope of Fig. 2 becomes confocal when a pinhole blocks light from all parts of the sample outside the focus. microscopes are used so often for conventional (thin sections) fluorescence microscopy applications. One thing our confocal microscope cannot do is look through walls. By that I mean that if a layer absorbs light, then deeper layers will be harder to see. That drop-off of visibility limits the sample thickness to about 50 /xm in many cases, although there are many instances of looking 0.5 mm into tissue. Point-Spread Function Now I want to discuss the physics of the effects just observed. In Fig. 2 we saw that light from a point source is imaged inside the sample. In the sample, that light forms a double cone, as shown in Fig. 5a. Figure 5b uses gray scale to show where the light is most intense. The scale is logarithmic so that the peak is 10 5 times brighter than the darkest areas. On a linear scale, shown in Fig. 5c, only the peak has any intensity. The cross section of the cone, shown as lines in the gray scale images, represents a numerical [ 1 ] THEORETICAL BASIS 7 Illuminated point in sample - ._- _;. _ I 1'-o ." - _- " - . . -" '_- o . . o . o -o . FIG. 4. Another schematic view of the confocal microscope. The point of interest is imaged in the pinhole, while light from the more proximate point is largely blocked by the pinhole. This organization is called "optical sectioning." a b c PSF: linear gray scale, NA = 0.65 PSF: log gray scale, NA = 0.65 40 p.m lateral 40 fun lateral FIG. 5. (a) Light from an objective lens fills a (double) cone. (b) The actual light intensity is plotted as a linear gray scale. The same presentation, with a logarithmic gray scale, is shown (c), with the lightest value being 10 5 times the darkest. [...]... the lateral resolution in terms of numerical aperture and wavelength for the confocal microscope The quantity Ar is the full width at half-maximum intensity of the confocal point-spread function I give this measure because there is some confusion in the literature as to how to use the Rayleigh criterion for resolution in the confocal situation Equation (6) gives the axial resolution (the optical section)... fluorescence The confocal microscope requires that the pinhole be optically conjugate to the illuminated spot in the thick sample That means that both the point source and the pinhole have to be imaged at the same place Such a confocal arrangement is possible if the microscope objective is highly achromatic or if the pinhole position is adjusted to compensate for chromaticity Some confocal microscopes... placed in front of the detectors Much of the cost of confocal microscopes has to do with changing those filters, the pinhole size, the choice of detector, the size of the scan (the field of view), and other parameters necessary to a useful picture Varieties of Confocal Microscope One of the engineering details I have been ignoring is the scanning engine Confocal microscopes come in two versions, O and... No pinhole is needed [ 11 THEORETICALBASIS 17 Light S o u r c e s Laser Real confocal microscopes have a lot of engineering details In general, these are not appropriate for this discussion, but one of the details i s - - t h e light source Confocal microscopes of the CM-P flavor use lasers as their source Disk-scanning confocal microscopes (CM-O) have the advantage of being able to use almost any... emerges in a tightly collimated coherent beam What that means for a confocal microscope is that the point source is very nearly perfect Lasers are also monochromatic and coherent, but the coherence is rather incidental for the use we make of the laser Coherent light is light whose waves are all exactly in phase That can be useful in confocal microscopy, but it is not necessary Speckle In fact, coherent light... microscope Bleaching One of the major uses of confocal microscopy is for fluorescence imaging The fluorophores used tend to bleach out when exposed to too much light Bleaching is generally thought to be proportional to light dose, although there are some examples of nonlinearities, both favorable and unfavorable to the high intensities of the laser-scanning confocal microscopes 1 Fluorophore saturation... Excitation 21 L i g h t P a t h s for E m i s s i o n Illuminating Aperture aroic 1TOt lbjective Lel Focal Plane cCoell or/ versiip In-focus Rays Out-of-focus Rays FIG 1 The confocal principle in epifluorescence scanning confocal microscopy Light from laser source passes through the illuminating aperture, is reflected by the dichroic mirror, and is focused on one point in the specimen (left) After excitation,... information can be lost in the transition from numerical data to human perception A confocal microscope does not produce a planar image, it produces a three-dimensional array of sample values or voxels, which is even harder to present to the eye in a meaningful form Much of the image processing associated with confocal microscopy is devoted to overcoming the problem; it is visualization software, dedicated... need good apochromats or similar objectives Other confocal microscopes separate their colors before the pinholes, so each pinhole can be adjusted separately and less expensive objectives can be used This multipinhole design risks misalignment by a factor of the number of pinholes (usually three) There are trade-offs in both price and convenience Most confocal microscopists want the option of exciting... a point pinhole It turns out that we can use a pinhole that is about three resels across and still get almost all of the confocal effects 1,z An even bigger pinhole will blur the point-spread function enough to degrade the optical sectioning and contrast enhancement So my ideal confocal microscope will use a three resel pinhole, i.e., a pinhole that is three times the size of the Airy disk Magnification . Pawley, ed., in "Handbook of Biological Confocal Microscopy, " 3rd ed. Plenum, New York 1996. 2 T. Wilson, ed., in " ;Confocal Microscopy. " Academic Press, London, 1990 142-8555, Japan [ 11 THEORETICAL BASIS 3 [1] Theoretical Basis of Confocal Microscopy By ROBERT H. WEBB A Simple View A confocal microscope is most valuable in seeing clear images inside. inside the object or sample, and a pinhole detector, all three confocal with each other. That is a confocal microscope.l-3 This confocal microscope has all the features we need for looking at