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Confocal Microscopy PDF

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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 ton ew 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. LEAHCIM NNOC iiix 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 ALBERICO BORGHETrI (20), Department of Pathology, University of Wisconsin, Madi- Medicine, Clinical Nephrology, and Health son, Wisconsin 53706 Sciences, University of Parma, 43100 SILVIA M. ARRIBAS (15), Departamento de Parma, Italy Facultad de Fisiologia, Medicina, Universi- DAVID N. BOWSER (25), Confocal and Fluo- dad Aut6noma de Madrid, 28029 Madrid, rescence Imaging Group, Department of Spain Physiology, The University of Melbourne, Parkville, Victoria Australia 3052, GEORGE F. BABCOCK (18), Departments of Biology, and University Cell Surgery of Cin- DANIEL BROTCHIE (28), Unit of Ophthalmol- cinnati College of Medicine, and Shriners ogy, Department of Medicine, University of Hospitals for Cincinnati Children, BuIrnn-s Liverpool, Liverpool L69 3GA, United stitute, Cincinnati, Ohio 8550-76254 Kingdom WERNER BASCHONG (11), M. E. Insti- Miiller BUEHLER CHRISTOF (29), Department of Me- chanical Engineering, Massachusetts Insti- tute for Structural Biology and Department tute of Technology, Cambridge, Massachu- of Surgery, Oral Biozentrum of the Univer- setts 93120 sity of Basel, CH-4056 Basel, Switzerland NICK CALLAMARAS (10), Department of Neu- MIGUEL BERRIOS (4), Department of Pharma- robiology and Behavior, University of -ilaC cological Sciences and University Micros- fornia, Irvine, California 0554-79629 copy Imaging Center, University Hospital State University Center, Medical and of New SILVANO CAPITANI (12), Institute of Human Anatomy, University of ,ararreF Fer- 44100 York, Stony Brook, New York 8808-49711 rata, Italy KANTI D. BHOOLA (22), Department of Exper- H. DWIGHT CAVANAGH (14), Department of imental and Pharmacology, Faculty Clinical Ophthalmology, University of South- Texas of Medicine, University of Natal, Congella western Medical Center, Dallas, Texas ,1004 South Africa 7509-53257 MIKE BIRCH (28), Unit of Ophthalmology, CATERINA CINTI (12), Institute of Citomorfo- Department of Medicine, University of logia Normale e Patologica, C.N.R., 66100 Liverpool, Liverpool L69 3GA, United Chieti, Italy Kingdom DAVID E. COLFLESH (4), University Micros- GHASSAN BKAILY (8), MRCC Group in Ira- copy Imaging Center, University Hospital ralucsavoidraC-DnUor Depart- Interactions, University State Center, Medical and of New ment of Anatomy Biology, and Cell Faculty York, Stony Brook, New York 8808-49711 of Medicine, University of Sherbrooke, KIMBERLY A. CONLON (4), Department of Sherbrooke, Canada QuEbec, JIH 5N4 Pharmacological Sciences, University Hos- LOTHAR A. BLATTER (16), Department of pital and Medical Center, State University Physiology, Loyola University of ,ogacihC of New York, Stony Brook, New York Maywood, Illinois 35106 1568-49711 MATIqqIAS BOHNKE (30), Department of GuY Cox (3), Microscope Electron Uni- Unit, Ophthalmology, University of Bern, 3010 versity of Sydney, Sydney, New South Wales Bern, Switzerland ,6002 Australia ix X CONTRIBUTORS TO VOLUME 307 DANIEL CULLEN (34), Labo- Products Forest C. HOWARD VYVYAN (28), Department of -eF ,yrotar Department U.S. of For- Agriculture lat Infant Toxico-Pathology, and ytisrevinU est ,ecivreS Madison, Wisconsin 50735 of Liverpool, Liverpool L69 3GA, detinU Kingdom CRAIG J. DALY (15), Autonomic Physiology ,tinU University of ,wogsalG Glasgow 21G DAVID N. HOWELL (32), Departments of -aP 8QQ, Scotland, Kingdom United thology and Laboratory Medicine ,ecivreS snareteV Affairs Medical ,retneC Durham, MARKUS DI~RRENBERGER (11), Interdivi- North aniloraC ,50772 and Duke ytisrevinU Biozentrum Microscopy, Electron sional of Medical ,retneC Durham, North aniloraC eht University of Basel, CH-4056 Basel, 01772 Switzerland JoN R. INGLEEIELD (26), Di- Neurotoxicology RALE ENGELMANN (31), Leibniz-Institut for ,noisiv and Environmental Health National Neurobiology, D-39118 Magdeburg, Ger- Laboratory, Research Effects .S.U Environ- many Agency, Research Protection mental -nairT MARGHERITA FONTANA (27), Oral Biology elg Park, North Carolina 11772 Research Health and Oral ,etutitsnI Indiana HIROSHI ISHIKAWA (6), Department of Anat- ytisrevinU School of Indianapo- Dentistry, omy, Jikei School The University of Medi- ,sil Indiana 20264 ,enic Minato-ku, Tokyo ,1648-501 Japan RITA GATrI (20), Institute of Histology and DANIELLE JACQUES (8), MRCC Group ni -arI Embryology, General ytisrevinU of ,amraP ralucsavoidraC-Dnuor ,snoitcaretnI Depart- 00134 Parma, Italy ment of Anatomy Faculty Biology, and Cell GIAN CARLO GAZZOLA (20), Institute of Gen- of Medicine, University of Sherbrooke, Pathology, University eral of Parma, 00134 Sherbrooke, Quebec, Canada J1H 5N4 Parma, Italy JAMES V. JESTER (14), Department of Oph- CARLOS GONZ,~EZ-CABEZAS (27), Biol- Oral thalmology, ytisrevinU of Southwest- Texas In- Institute, Research Health and ogy Oral nre Medical Dallas, Center, Texas -53257 ytisrevinU anaid School of ,yrtsitneD India- 7509 ,silopan Indiana 20264 MANABU KAGAYAMA (5), Department of ENRICO GRATTON (29), Laboratory for Flu- Anatomy, Tohoku University School of orescence Dynamics, Department of ,yrtsitneD Sendai ,5758-089 Japan Physics, University of Illinois at Urbana- KI HEAN KIM (29), Department of lacinahceM ,ngiapmahC ,anabrU Illinois 10816 Engineering, Massachusetts Institute of IAN GRIERSON (28), Unit of Ophthalmology, Technology, Cambridge, Massachusetts Department of Medicine, University of 93120 Liverpool, Liverpool L69 3GA, United SUSAN M. KNOBEL (21), Department of Mo- Kingdom ralucel Biophysics, and Physiology -rednaV HASHIMOTO HISASHI (6), Department of Anat- bilt ,ytisrevinU Nashville, Tennessee 23273 omy, Jikei The School University of Medi- SAMUEL KO (2), Department of ,yrtsimehcoiB cine, Minato-ku, Tokyo ,1648-501 Japan, The Chinese University of Hong Kong, and Center for Biogenic ,secruoseR In- The ,nitahS N.T., Hong Kong stitute of Physical and Chemical hcraeseR (RIKEN), Tsukuba, Ibarald ,4700-503 S. K. KONG (2), Department of ,yrtsimehcoiB napaJ The Chinese University of Hong Kong, ,nitahS N.T., Hong Kong PENNY HOGG (28), Unit of Ophthalmology, Department of Medicine, University of ULRICH KCEHCSTIBUK (13), Institut far Medi- Liverpool, Liverpool L69 3GA, United Physik zinische und Biophysik, t~itisrevinU Kingdom ,retsnitM Germany Mtinster, D-48149 CONTRIBUTORS TO VOLUME 307 xi THORSTEN KUES (13), Institut far Medizin- LUCA M. NERI (12), Institute of Human ische Physik und Biophysik, Universitat Anatomy, University of ,ararreF 44100 Fer- Miinster, D-48149 Miinster, Germany ,arar Italy MORIAKI KUSAKABE (6), Center for Biogenic HISAVUKI OHATA (24), Department of Phar- Resources, The Institute of Physical and macology, School of Pharmaceutical Sci- Chemical Research (RIKEN), Tsukuba, ences, Showa University, Shinagawa-ku, Ibaraki ,4700-503 Japan Tokyo ,5558-241 Japan C. Y. LEE (2), Department of Biochemistry, GUIDO ORLANDINI (20), Department of Clini- The Chinese University of Hong Kong, cal Medicine, Nephrology, and Health Sci- Shatin, N.T., Hong Kong ences, University of Parma, 43100 Parma, Italy P. Y. Lui (2), Department of Biochemistry, The Chinese University of Hong Kong, IAN PARKER (10), Department of Neurobiol- Shatin, N.T., Hong Kong ogy and Behavior, University of ,ainrofilaC Irvine, California 0554-79629 ANNA MANDINOVA (11), M. E. Miiller Insti- tute for Structural Biology, Biozentrum of REINER PETERS (13), Institutfar Medizinische the University of Basel, CH-4056 Basel, Physik und Biophysik, t~itisrevinU Miinster, Switzerland D-48149 MUnster, Germany CARLOS B. MANTILLA (17), Mayo Clinic, W. MATI'HEW PETROLL (14), Department of Rochester, Minnesota 50955 Ophthalmology, University of Texas South- western Medical Center, Dallas, Texas FRANCESCO A. MANZOLI (12), Institute of 7509-53257 Human Anatomy, University of Bologna, 62104 Bologna, Italy STEVEN PETROU (25), Confocal and Fluores- cence Imaging Group, Department of Phys- NADIR M. MARALDI (12), Institute of Cito- iology, The University of Melbourne, Park- morfologia Normale e Patologica, C.N.R., ,eUiv Victoria 3052, Australia and Laboratory of Biologia Cellulare e Microscopia Elettronica, L O.R., 40136 DAVID W. PISTON (21), Department of Molec- Bologna, Italy, and Institute of Human ular Physiology and Biophysics, Vanderbilt Anatomy, University of Bologna, 40123 University, Nashville, Tennessee 23273 Bologna, Italy TORSTEN PORWOL (7), Max-Planck-Institut BARRY R. MASTERS (29, 30), Department of far Molekulare Physiologie, D-44202 Dort- Mechanical Engineering, Massachusetts In- mund, Germany stitute of Technology, Cambridge, Massa- PIERRE POTHIER (8), MRCC Group in Ira- chusetts ,93120 and Department of Ophthal- ralucsavoidraC-Dnuor Interactions, Depart- mology, University of Bern, 3010 Bern, ment of Anatomy and Cell Biology, Faculty Switzerland of Medicine, University of Sherbrooke, JOHN C. MCGRA77-I (15), Autonomic Physiol- Sherbrooke, QuEbec, Canada J1H 5N4 ogy Unit, University of Glasgow, Glasgow Y. S. PRAKASH (17), Mayo Clinic, Rochester, G12 8QQ, Scotland, United Kingdom Minnesota 50955 SARA E. MILLER (32), Departments of Micro- DESHANDRA M. RAIDOO (22), Department of biology and Pathology, Duke University Experimental and Clinical Pharmacology, Medical Center, Durham, North Carolina Faculty of Medicine, University of Natal, 01772 Congella 4001, South Africa KAZUTAKA MOMOSE (24), Department of GOUSEI RIE (24), Department of Pharmacol- Pharmacology, School of Pharmaceutical ogy, School of Pharmaceutical Sciences, Sciences, Showa University, Shinagawa-ku, Showa University, Shinagawa-ku, Tokyo Tokyo ,5558-241 Japan ,5558-241 Japan xii CONTRIBUTORS TO VOLUME 307 NElL ROBERTS (28), Re- Resonance Magnetic STEFANO SQUARZONI (12), Institute of -otiC hcraes ,ertneC ytisrevinU of Liv- Liverpool, morfologia Normale e ,acigolotaP C.N.R., erpool L69 3GA, Kingdom United 63104 Bologna, Italy RONDA NICOLETTA (20), Department of -inilC GEORGE K. STOOKEY (27), Oral Biology and lac ,enicideM Nephrology, and Health Sci- Oral Health Research Institute, Indiana ,secne University of Parma, 43100 ,amraP ytisrevinU School of ,yrtsitneD Indianapo- ylatI ,sil Indiana 20264 BERNHARD A. SABLE (31), Institute of Medi- ANJA-ROSE REIAMHORTS (7), Nikon GmbH, lac Psychology, Otto-v.-Guericke Univer- 27404-D Dasseldorf Germany sity of Magdeburg, D-39120 ,grubedgaM LIBORIO STUPPIA (12), Institute of Biologia e Germany Genetica, University .G" d'Annunzio" OCATRAPS SANTI (12), Institute of Citomorfo- 00166 ,iteihC Italy aigol Normale e ,acigolotaP C.N.R., 00166 ,iteihC Italy SUETFERLIN ROSMARIE (11), .M .E Mallet In- stitute for Biology, Structural Biozentrum SASANO YASUYUKI (5), Department of Anat- of eht University of Basel, CH-4056 Ba- omy, Tohoku University School of Den- ,les Switzerland ,yrtsit Sendai ,5758-089 Japan XUEJUN SUN (9), Department of ,ygolocnO ROCHELLE D. MOOLB-ZTRAWHCS (26), -eD ytisrevinU of Alberta, Cross Cancer In- partment of Bi- and Pharmacology Cancer ,etutits Edmonton, Alberta T6G 1Z2, Can- ology, Duke University Medical ,retneC ada Durham, North Carolina 01772 YOSUKE UJIKE (24), Department of Pharma- AKIHISA SEGAWA (19), Department of Anat- ,ygoloc School of lacituecamrahP ,secneicS omy, School of Medicine, Kitasato Uni- Showa University, Shinagawa-ku, Tokyo ,ytisrev Sagamihara, Kanagawa ,5558-822 ,5558-241 Japan napaJ GARY C. SIECK (17), Mayo ,cinilC ,retsehcoR ALAIN NEHCSSALPREDNAV (33), Immunol- Minnesota 50955 ogy-Vaccinology, Faculty of yranireteV Medicine, University of Liege, B-4000 GEOFFREY L. SMITH (33), Sir William Dunn ,egOiL Belgium School of Pathology, University of ,drofxO Oxford OX1 3RE, Kingdom United ROBERT H. WEBB (1), Eye Schepens hcraeseR ,etutitsnI Laboratories Wellman and of Pho- CELIA J. NAMYNS (22), Department of Experi- ,enicidemot Hospi- General Massachusetts and mental lacinilC Faculty Pharmacology, ,lat Boston, sttesuhcassaM 41120 of ,enicideM University of Natal, allegnoC ,1004 South Africa DAVID ALAN WILLIAMS (25), Confocal and PETER T. C. So (29), Department of -inahceM Fluorescence Imaging Group, Depart- lac Engineering, sttesuhcassaM Institute of ment of Physiology, The University of Technology, Cambridge, Massachusetts Melbourne, Parkville, 3052, Victoria Aus- 93120 ailart RUSSELL N. SPEAR (34), Department of Plant KAZUHIRO YAMAGUCHI (23), Department of Pathology, University of Madi- Wisconsin, ,enicideM School of ,enicideM Keio -revinU son, Wisconsin 60735 sity, Tokyo ,2858-061 Japan EBERHARD SPIESS (7), Biomedizinische MASAYUKI OTOMAMAY (24), Department of Strukturforschung, Deutsches Krebs- Pharmacology, School of lacituecamrahP forschungszentrum, D-69009 ,grebledieH ,secneicS Showa ,ytisrevinU ,uk-awaganihS ynamreG Tokyo ,5558-241 Japan [ 11 LACITEROEHT SISAB 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., ni "Handbook of Biological Confocal Microscopy," 3rd ed. Plenum, New York 1996. 2 T. Wilson, ed., ni "Confocal Microscopy." Academic Press, London, 1990. 3 R. H. Webb, Rep. Prog. Phys. 59, 427 1996. © Copyright 9991 by Press Academic All of rights reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 703 99/9786-6700 00.03$ 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, thisd evice also discriminateasg ainst 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 LACITEROEHT SISAB 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. 5'4 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). 6 THEORY AND PRACTICAL CONSIDERATIONS [ 11 Image of illuminated sample point ~ . ~ I Detector (PMT) [ is lla that gets through eht pinhole thg source \ ~-- Brightly illuminated .~point ni sample noitanimullI light path Collection light path .GIF .3 The microscope of confocal 2 becomes Fig. when from light a blocks pinhole lla parts of the outside the sample .sucof 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.

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