Table Of ContentMETHODS IN CELL
BIOLOGY
Prepared under the Auspices of the American Society for Cell Biology
VOLUME 30
Fluorescence Microscopy of Living Cells in Culture
AErt B. Quantitative Fluorescence
Microscopy-Imaging and Spectroscopy
Edited by
D. LANSING TAYLOR
DEPARTMENT O F BIOLOGICAL SCIENCES
CENTER FOR FLUORESCENCE RESEARCH IN BIOMEDICAL SCIENCES
CARNEGIE-MELLON UNIVERSITY
PITTSBURGH, PENNSYLVANIA
YU-LI WANG
CELL BIOLOGY GROUP
WORCESTER FOUNDATION FOR EXPERIMENTAL BIOLOGY
SHREWSBURY. MASSACHUSETTS
ACADEMIC PRESS, INC.
Hnrcourt Brace Jowoovich. Publishers
San Diego New York Berkeley Boston
London Sydney Tokyo Toronto
Series Editor
LESLIE WILSON
Departmenr of Biological Sciences
University of California, Santa Barbara
Santa Barbara, California
IAS C B I
COPYRIGHT @J 1989 BY ACADEMICP RESS, INC.
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8 9 9 0 9 1 9 2 9 8 7 6 5 4 3 2 1
Multiple spectral parameter mapping image created by the simultaneous display of three individual
images (See Chapter 17).
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
DAVID A. AGARDH, oward Hughes Medical L. ERNSTD, epartment of Biological Sciences,
Institute and Department of Biochemistry, Center for Fluorescence Research in the
University of California, San Francisco, San Biomedical Sciences, Carnegie Mellon Univer-
Francisco, California 94143 (353) sity, Pittsburgh, Pennsylvania 15213 (449)
DONNAJ . ARNDT-JOVIN, Department O f G. W. FISHER,D epartment of Biological
Molecular Biology, Max Planck Institute for Sciences, Center for Fluorescence Research in
Biophysical Chemistry, D-3400 Gottingen, the Biomedical Sciences, Carnegie Mellon Uni-
Federal Republic of Germany (417) versity, Pittsburgh, Pennsylvania 15213 (157)
Davm GROSSD,e partment of Biochemistry and
DANIEAL XELRODB,i ophysics Research Division Program in Molecular and Cellular Biology,
and Department of Physics, University of University of Massachusetts, Amherst,
Michigan, Ann Arbor, Michigan 48109 (246, Massachusetts 01003 (193)
333, 399)
EDWARHD. HELLENB,i ophysics Research Divi-
sion and Department of Physics, University
G. J. BRAKENHOFDFe,p artment of Electron
of Michigan, Ann Arbor, Michigan 48109
Microscopy and Molecular Cytology, Uni-
(399)
versity of Amsterdam, 1018 TV Amsterdam,
The Netherlands (379)
BRIAN HERMANL,a boratories for Cell Biology,
Department of Cell Biology and Anatomy,
G. R. BRIGHTD, epartment of Biological University of North Carolina School of
Sciences, Center for Fluorescence Research in Medicine, Chapel Hill, North Carolina 27599
Biomedical Sciences, Carnegie Mellon Univer- (220)
sity, Pittsburgh, Pennsylvania 15213 (157,449)
YAsusHi HiruoKa, Howard Hughes Medical
JOSEPH BRYAND, epartments of Medicine and Institute and Department of Biochemistry,
Cell Biology, Baylor College of Medicine, University of California, San Francisco, San
Houston, Texas 77030 (48) Francisco, California 94143 (353)
SHiNyA I N O L ~M, arine Biological Laboratory,
P. CONRADD, epartment of Biological Sciences,
Woods Hole, Massachusetts 02543 (85)
Center for Fluorescence Research in the
Biomedical Sciences, Carnegie Mellon Univer-
~ E L J K OJ ERIL.evit:, Rugjer Boskovic Institute,
sity, Pittsburgh, Pennsylvania 15213 (449)
Zagreb, Croatia, Yugoslavia (48)
R. DEBlAslo, Department of Biological THOMAMS . JOVIN, Department of Molecular
Sciences, Center for Fluorescence Research in Biology, Max Planck Institute for Biophysical
the Biomedical Sciences, Carnegie Mellon Uni- Chemistry, D-3400 Gottingen, Federal
versity, Pittsburgh, Pennsylvania 15213 (449) Republic of Germany (417)
ELLIOT L. ELSON, Department of Biological LESLIE M. LOEW,D epartment of Physiology,
Chemistry, Washington University School of University of Connecticut Health Center,
Medicine, St. Louis, Missouri 63110 (307) Farmington, Connecticut 06032 (193)
xi
xii CONTRIBUTORS
N. NANNINGAD, epartment of Electron LOUISC . SMITH,D epartment of Medicine,
Microscopy and Molecular Cytology, Uni- Baylor College of Medicine, Houston, Texas
versity of Amsterdam, 1018 TV Amsterdam, 77030 (48)
The Netherlands (379)
D. TAYLORD, epartment of Biological Sciences,
Center for Fluorescence Research in the
M. NEDERLOFD, epartment of Biological
Sciences, Center for Fluorescence Research in Biomedical Sciences, Carnegie Mellon Univer-
sity, Pittsburgh, Pennsylvania 15213 (449, 157)
the Biomedical Sciences, Carnegie Mellon Uni-
versity, Pittsburgh, Pennsylvania 15213 (449)
RWER Y. TSIEN,D epartment of Physiology-
Anatomy, University of California, Berkeley,
HONG QIAN, Department of Biological
California 94720 (127)
Chemistry. Washington University School of
Medicine, St. Louis, Missouri 63110 (307)
H. T. M. VAN DER VOORT,D epartment of
Electron Microscopy and Molecular Cytology,
J . ROGOWSKAD, epartment of Biological
Sciences, Center for Fluorescence Research in University of Amsterdam, 1018 TV Amster-
dam, The Netherlands (379)
the Biomedical Sciences, Carnegie Mellon Uni-
versity, Pittsburgh, Pennsylvania 15213 (157)
E. A. VAN SPRONSEND,e partment of Electron
K. RYAND, epartment of Biological Sciences, Microscopy and Molecular Cytology, Uni-
Center for Fluorescence Research in the versity of Amsterdam, 1018 TV Amsterdam,
Biomedical Sciences, Carnegie Mellon Univer- The Netherlands (379)
sity, Pittsburgh, Pennsylvania 15213 (449)
A. WAGGONERD,e partment of Biological
JOHNW . SEDAT,H oward Hughes Medical Sciences, Center for Fluorescence Research in
Institute and Department of Biochemistry, and the Biomedical Sciences, Carnegie Mellon Uni-
Biophysics, University of California, San versity, Pittsburgh, Pennsylvania 15213 (449)
Francisco, San Francisco, California 94143
BRENTW IESE,D epartments of Medicine and
(353)
Cell Biology, Baylor College of Medicine,
Houston, Texas 77030 (48)
PETERS HAWH, oward Hughes Medical Institute
and Department of Biochemistry, University
of California, San Francisco, San Francisco, DAVIDE . WOLF, Worcester Foundation for
California 94143 (353) Experimental Biology, Shrewsbury, Massachu-
setts 01545 (271)
JESSEE . SISKEND, epartment of Microbiology
and Immunology, College of Medicine, Uni- IANT . YOUNGD, epartment of Applied Physics,
versity of Kentucky, Lexington, Kentucky Delft University of Technology, Delft, The
40536 (113) Netherlands (2)
PREFACE
Fluorescence techniques are uniquely suitable for probing living cells
because of their sensitivity and specificity. Since fluorescence from a single
cell can be detected with a microscope both as an image and as a photometric
signal, fluorescence microscopy has great potential for qualitative and quan-
titative studies on the structure and function of cells. However, owing to
previous technical limitations, fluorescence has been used primarily for stain-
ing fixed cells for many years. It has not been until recently that the true power
of the techniques has evolved for use with single living cells. The most
important advances that have made this possible include the development of
(1) probes for specific structures or environmental parameters; (2) methods
for delivering fluorescent probes into living cells; (3) methods for detecting
weak fluorescence signals from living cells; and (4) methods for acquiring,
processing, and analyzing fluorescence signals with microscopes.
The primary purpose of this and the accompanying volume of Methods
in Cell Biology is to provide readers with detailed descriptions of methods
in these four areas. While techniques for flourescence spectroscopy in solu-
tion are described in various sources, there has been no convenient source
for the methods specifically applied to living cells. Even with an extensive
literature search, one often finds crucial technical details, including instnunen-
tation, sample handling, and precautions, left out in many research articles.
It is our hope that these volumes will provide enough detail to make the new
developments approachable by most investigators. Although some biological
perspectives are provided in many chapters, the main emphasis of the volumes
is practical laboratory methods; the job of biological reasoning and experi-
mental design is left to individual investigators. The books are thus targeted
primarily at experienced cell biologists who wish to apply modem fluorescence
techniques. However, they should also be of great interest to biochemists and
molecular biologists who attempt to correlate results in test tubes with activities
in living cells. In addition, many chapters should be valuable to those
specializing in instrumentation, including microscopy, electronic imaging, and
digital image processing.
The two volumes represent a collective effort of many investigators. The
chapters were assembled by specific areas which, in our view, were important
or held great promise in the future. We then invited those researchers with
extensive experience in the particular area to make contributions. There was
a certain degree of subjectiveness in choosing the topics. On the one hand,
we have included topics crucial to, but not specific for, fluorescence microscopy
of living cells, including microscopy cell culture, microinjection, microscopy
xiii
xiv PREFACE
photometry, and low light level imaging. On the other hand, we decided to
sacrifice several useful topics that were either not in a mature stage of develop-
ment or where we were unable to obtain a commitment from an authority.
The first volume (Volume 29) deals with the preparation, delivery, and detec-
tion of fluorescent probes. The first half is focused on the preparation of
specific structural probes, including fluorescent analogs that can be utilized
by living cells in structural assembly, fluorescent molecules that bind to specific
cellular components, and probes that can be used to label particular cellular
compartments. There are special challenges in the preparation of each class
of probes, including proteins, small peptides, heterocyclic compounds, lipids,
and polysaccharides. Subsequent chapters discuss factors that determine the
destination of probes and methods for delivering probes to specific sites in
living cells. The second half of the first volume discusses the detection of
fluorescent probes in living cells, including issues related to sample physiology
(microscopy cell culture), optics (basic fluorescence microscopy), and signal
detection (electronic photometry and imaging, immunoelectron microscopic
detection of fluorophores). The last few chapters introduce modern techni-
ques in image detection and provide a continuity to quantitative analytical
methods covered in Volume 30.
The second volume (Volume 30) explores a combination of the theoretical
and technical issues related to the quantitation of fluorescence signals in the
living cell with a light microscope. The first section explores the engineering
principles required in the characterization of the performance of an imaging
system. The use of system validation procedures and quantitative fluorescent
standards are explored in detail. The remainder of Volume 30 is devoted to
specific applications and optical methods. A mix of theoretical and practical
issues is discussed, including the measurement of membrane potential, ionic
concentrations, tracer diffusion coefficients, total internal reflection, fluores-
cence polarization, and three-dimensional reconstruction. Thus, the two-
volume set defines a technical continuum from organic chemistry, through
biochemistry, cell biology, physics, and engineering, to computer science. The
present status of the field reflects the occurrence of a revolution in cell
biological research.
We would like to thank all contributing authors for providing us with their
extensive experience in various areas. Most of them have worked closely with
us in planning their chapters and minimizing overlaps, then submitting
excellent manuscripts in a timely fashion and answering questions which arose
during editing.
D. LANSINGTA YLOR
Yu-LI WANG
Chapter 1
Image Fidelity: Characterizing the Imaging
Transfer Function
IAN T. YOUNG
Depdment of Applied Pbysia
Delft Univenity of Technology
Delft, Tbe Netberlandr
I. Introduction
A. The Reality of Distortion
B. Models of a Fluorescence Imaging System
11. The Concept of a Linear, Shift-Invariant (LSI) System
A. What Is Linearity?
B. What Is Shift Invariance?
C. Is a Fluorescence Imaging System LSI?
D. Fluorescence Image as a Superposition Result-Convolution
111. Characterizing LSI Systems with Sinusoids
A. Sinusoids in/Sinusoids out
B. The Complex Sinusoid and Convolution
C. Description of an Image in Terms of Complex Sinusoids-the Fourier
Representation
IV. Characterizing the Fluorescence Imaging System
A. Model of a Fluorescence Imaging System (Reprise)
B. Role of Microscope “Parameteys”
C. The Fundamental Lens Transfer Function H(o,)-the OTF
D. The Effect of Magnification
E. The Total OTF
F. Other Possible Effects
V. The Contrast Modulation Transfer Function (CMTF)
A. The Definition
B. The Measurement
VI. Relating the CMTF and the OTF
A. TheTheory
B. Practical Implementation
VII. Implications
A. The Resolving of Fine Detail
1
Copyright 0 1989 by Academic Press, Inc.
METHODS IN CELL BIOLOGY. VOL. M All rights of reproduction in any form reserved.
