Confocal Microscopy

669 Pages·1999·11.7 MB·English

by  Conn P.M.

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.