Fluorescence Microscopy of Living Cells in Culture Part B. Quantitative Fluorescence Microscopy—Imaging and Spectroscopy PDF
Preview Fluorescence Microscopy of Living Cells in Culture Part B. Quantitative Fluorescence Microscopy—Imaging and Spectroscopy
METHODS 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. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS. ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM. WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC . San Diego. California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road. London NWI 7DX LIBRARYO F CONGRESS CATALOG CARD NUMBER:6 4-14220 ISBN 0-12-564130-3 (alk. paper) PRINTED IN THE UNITED STATES OF AMERICA 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.
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