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Fluorescence Microscopy and Fluorescent Probes
Fluorescence Microscopy and Fluorescent Probes
Edited by
Jan Slavik Czech Academy of Sciences
Prague, Czech Republic
Springer Science+Business Media, LLC
Library of Congress Catalog1ng-in-Publication Data
Fluorescence microscopy and fluorescent probes / edited by Jan Slavik. p. cm.
"Based on the proceedings of the conference on Fluorescence Microscopy and Fluorescent Probes, held June 25-28, 1995, in Prague, Czech Republic"—T.p. verso.
Includes bibliographical references and index. ISBN 978-1-4899-1868-0 1. Fluorescence microscopy—Congresses. 2. Fluorescent probes-
-Congresses. I. Slavik, Jan, Ph.D. QH212.F55F55 1996 578—dc20 96-43731
CIP
Based on the proceedings of the conference on Fluorescence Microscopy and Fluorescent Probes, held June 25-28, 1995, in Prague, Czech Republic
ISBN 978-1-4899-1868-0 ISBN 978-1-4899-1866-6 (eBook) DOI 10.1007/978-1-4899-1866-6
© 1996 Springer Science+Business Media New York Originally published by Plenum Press, New York in 1996
Softcover reprint of the hardcover 1st edition 1996
All rights reserved
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No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise,
without written permission from the Publisher
CONTRIBUTORS
H. Acker Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
lolana T.P. Albrechtova Institute of Biology II, Botany Albert-Ludwigs-University Freiburg im Breisgau, Germany
Marcel Ameloot Limburgs Universitair Centrum Universitaire Campus Diepenbeek, Belgium
Robert E. Anderson Neurosurgical Research Laboratory Mayo Clinic Rochester, MN, USA
K. Autio VTT Biotechnology and Food Research Espoo, Finland
Vladimir Baltic Institute of Oncology Department of Experimental Oncology Sremska Kamenica, Yugoslavia
M. Bartunek Department of Genetics and
Microbiology Faculty of Natural Sciences Charles University Prague, Czech Republic
G. Bellomo Department of Medical Sciences University of Torino Novara, Italy
Mehdi Benchaib Cytologie Analytique University of Claude Berbnard Lyon, France
Rolf Bjerkvig University of Bergen Department of Anatomy and Cell Biology Bergen, Norway
loachim Bradl Institute of Applied Physics University of Heidelberg Heidelberg, Germany
lana Brezinova Institute of Hematology and Blood
Transfusion Prague, Czech Republic
Odd Terje Brustugun University of Bergen Department of Anatomy and Cell Biology Bergen, Norway
Paul-Andre Bryon Cytologie Analytique University of Claude Bernard Lyon, France
v
Petr Cimprich Institute of Physiology Czech Academy of Sciences Prague, Czech Republic
Robert M. Clegg Max Planck Institute for Biophysical
Chemistry Department of Molecular Biology G6ttingen, Germany
Christoph Cremer Institute of Applied Physics University of Heidelberg Heidelberg, Germany
Ben Crystall Department of Chemistry Imperial College London SW7 2AY, UK
Jean Davoust Centre d'Immunologie CNRSINSERM de Marseille-Luminy
Marseille, France
Richard Delorme Cytologie Analytique University of Claude Bernard Lyon, France
Denis Demandolx Centre d'Immunologie CNRSINSERM de MarseilIe-Luminy
Marseille, France
Barbora Denksteinova Institute of Physics Charles University Prague, Czech Republic
Tom Deutsch Wellman Laboratories of Photomedicine Department of Dermatology Harvard Medical School Massachusetts General Hospital Boston, MA 02114, USA
vi
Stein Ove Doskeland Department of Anatomy and Cell Biology University of Bergen Bergen, Norway
Ralph E. Durand B.C. Cancer Research Centre Vancouver, B.C., Canada
Ian Durrant Research and Development Amersham International Amersham, UK
MarkusDurm Institute of Applied Physics University of Heidelberg Heidelberg, Germany
I1ya V. Eigenbrot Department of Chemistry Imperial College London SW7 2AY, UK
R.I. Errington Oxford University Physiology Department Oxford OXI 3PT, UK
M.D. Fricker Oxford University Plant Sciences Department Oxford OXI 3RB, UK
Dana Gaskova Institute of Physics Charles University Prague, Czech Republic
Hans C. Gerritsen Debye Institute Department of Molecular Biophysics University of Utrecht Byus Ballot Laboratory Utrecht, The Netherlands
Bjorn Tore Gjertsen Department of Anatomy and Cell Biology, University of Bergen Bergen, Norway
Eva Grapengiesser Department of Medical Cell Biology Uppsala University Biomedicum Uppsala, Sweden
Martin Gregor Department of Physiology Faculty of Science Charles University Prague, Czech Republic
Michael H. Gschwend Institut fur Lasertechnologien in der
Medizin und MeBtechnik Universitat VIm, Ulm, Germany
Leslie Gubba Amersham Life Science Inc. Pittsburgh, P A, USA
Lars Guldfeldt Department of Dairy and Food Science Food Microbiology, The Royal Veterinary and Agricultural
University Frederiksberg C, Denmark
Heinz Gundlach Carl Zeiss Jena GmbH Division of Microscopy J ena, Germany
PetrHach Institute of Histology and Embryology 1st Medical Faculty Charles University Prague, Czech Republic
Tayyaba Hasan Wellman Laboratories of Photo medicine Department of Dermatology Harvard Medical School Massachusetts General Hospital Boston, MA 02114, USA
Michael Hausmann Institute of Applied Physics University of Heidelberg Heidelberg, Germany
Brian Herman Laboratories for Cell Biology Department of Cell Biology and
Anatomy University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
PetrHerman Institute ofphysics Charles University Prague, Czech Republic
MartinHof Department of Physical Chemistry Faculty of Natural Sciences Charles University Prague, Czech Republic
ZsoltHollo National Institute ofHaematology,
Blood Transfusion and Immunology Budapest, Hungary
Ales Holoubek Institute of Physics Charles University Prague, Czech Republic
Laszlo Homolya National Institute ofHaematology,
Blood Transfusion and Immunology Budapest, Hungary
Jan Jaap ter Horst Limburgs Universitair Centrum Universitaire Campus Diepenbeek, Belgium
Rudi Hutterer Department of Physical Chemistry University ofWiirzburg Wiirzburg, Germany
Seiichi Iinuma Wellman Laboratories of Photo medicine Department of Dermatology Harvard Medical School Massachusetts General Hospital Boston, MA 02114, USA
vii
Elzbieta Interewicz Department of Physiology Faculty ofpharmacy, Medical Academy Warsaw, Poland
Mogens Jakobsen Department of Dairy and Food Science Food Microbiology The Royal Veterinary and Agricultural
University Frederiksberg C, Denmark
Blanka Janderovci Department of Genetics and
Microbiology Faculty of Natural Sciences Charles University Prague, Czech Republic
Bruce G. Jenks Department of Animal Physiology Nijmegen Institute for Neurosciences Nijmegen, The Netherlands
Marie Jirkovskci Institute of Histology and Embryology 1st Medical Faculty Charles University Prague, Czech Republic
PetrKaren Institute of Physiology Czech Academy of Sciences Prague, Czech Republic
RolfK.H. Kinne Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
Ralf Kinscherf Department of Anatomy and Cell
Biology III University of Heidelberg Heidelberg, Germany
Christoph Kohler Department of Anatomy and Cell
Biology III University of Heidelberg Heidelberg, Germany
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Karsten Konig Institut fur Molekulare Biotechnologie Jena, Germany
Werner IH. Koopman Department of Animal Physiology Nijmegen Institute for Neurosciences Nijmegen, The Netherlands
Maarit Koskinen VTT Biotechnology and Food Research Espoo, Finland
Ivan Krekule Institute of Physiology Czech Academy of Sciences Prague, Czech Republic
Lucie Kubinovci Institute of Physiology Czech Academy of Sciences Prague, Czech Republic
Margaret E. Langmuir Covalent Associates, Inc. Woburn, MA 01801, USA
Lotte Lammert Department of Dairy and Food Science The Royal Veterinary and Agricultural
University Frederiksberg C, Denmark
Karen A. LeCompte Covalent Associates, Inc. Woburn, MA 01801, USA
Horst Ludwig Institute of Physical Chemistry University of Heidelberg Heidelberg, Germany
Jan MalinskY Institute ofphysics Charles University Prague, Czech Republic
Wolf Malkusch Image Analysis Division Kontron Elektronik GmbH Eching, Germany
Vera Maravic-Stojkovic Institute of Oncology Department of Experimental Oncology Sremska Kamenica, Yugoslavia
E.K. Matthews Department of Pharmacology University of Cambridge Cambridge, CB2 1QJ, UK
Gunnar Mellgren Department of Anatomy and Cell Biology University of Bergen Bergen, Norway
E. Merten Max-Planck-Institute for Molecular Physiology Dortmund, Germany
Jurgen Metz Department of Anatomy and Cell Biology University of Heidelberg Heidelberg, Germany
Fredric B. Meyer Neurosurgical Research Laboratory Mayo Clinic Rochester, MN, USA
K yra Michalova Third Medical Department General Hospital, Charles University Prague, Czech Republic
Vera Michalova Third Medical Department General Hospital, Charles University Prague, Czech Republic
Marianna Muller National Institute ofHaematology,
Blood Transfusion and Immunology Budapest, Hungary
Jose-Enrique O'Connor Departament de Bioquimica i
Biologia Molecular Facultad de Medicina Universitat de Valencia Valencia, Spain
Norbert Opitz Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
Izabela Orlanska Department of Physical Chemistry and
Department of Physiology Faculty of Pharmacy, Medical Academy Warsaw, Poland
Giuseppina Palladini Department of Internal Medicine IRCCS, Policlinico S.Matteo Pavia, Italy
Daniel Palous Institute of Histology and Embryology 1st Medical Faculty, Charles University Prague, Czech Republic
T. Parkkonen VTT Biotechnology and Food Research Espoo, Finland
Peter Pavlov Department of Anatomy and Cell
Biology III University of Heidelberg Heidelberg, Germany
D. Phillips Department of Chemistry Imperial College London SW7 2AY, UK
Jaromir Plasek Institute of Physics Charles University Prague, Czech Republic
Torsten Porwol Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
Jacek Przybylski Department ofphysical Chemistry and
Department of Physiology Faculty of Pharmacy, Medical Academy Warsaw, Poland
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D. Rickwood Biology Department University of Essex Colchester C04 3SQ, UK
Bernd Rinke Institute of Applied Physics University of Heidelberg Heidelberg, Germany
Eric W. Roubos Department of Animal Physiology Nijmegen Institute for Neurosciences Nijmegen, The Netherlands
Carlos P. Rubbi Biology Department, University of Essex Colchester C04 3 SQ, UK
Reinhard Sailer Institut fur Lasertechnologien in der Medizin und MeBtechnik
Universitat Ulm, Ulm, Germany
Balazs Sarkadi National Institute ofHaematology,
Blood Transfusion and Immunology Budapest, Hungary
Wim llM. Scheenen Department of Biomedical Sciences University of Pad ova Padova, Italy
Herbert Schneckenburger Institut fur Lasertechnologien in der Medizin und MeBtechnik
Universitat Ulrn, Ulrn, Germany
Bernhard Schneider Institute of Applied Physics University of Heidelberg Heidelberg, Germany
Peter C. Schneider Max Planck Institute for Biophysical
Chemistry Department of Molecular Biology Gottingen, Germany
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Kevin T. Schomacker Wellman Laboratories of Photomedicine Department of Dermatology Harvard Medical School Massachusetts General Hospital Boston, MA 02114, USA
Henrik Siegumfeldt Department of Dairy and Food Science Food Microbiology The Royal Veterinary and Agricultural University
Frederiksberg C, Denmark
Karel Sigler Institute of Microbiology Czech Academy of Sciences Prague, Czech Republic
Jan Slavik Institute of Physiology Czech Academy of Sciences Prague, Czech Republic
Catherine Souchier Cytologie Analytique University of Claude Bernard Lyon, France
Paul Steels Limburgs Universitair Centrum Universitaire Campus Diepenbeek, Belgium
Wolfgang S.L. StrauB Fachhochschule Aalen Optoelektronik Aalen, Germany
T. M. Sundt Jr. Neurosurgical Research Laboratory Mayo Clinic Rochester, MN, USA
Jan Tachezy Department of Parasitology Faculty of Science Charles University Prague, Czech Republic
Anders Tengholm Department of Medical Cell Biology Uppsala University Biomedicum Uppsala, Sweden
B.R. Terry BioImage:S Novo Nordisk Research Satellite Soborg, Denmark
Hanna Tinel Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
Claus Usinger Department of Anatomy and Cell Biology III University of Heidelberg Heidelberg, Germany
L.Valasek Department of Genetics and
Microbiology Faculty of Natural Sciences Charles University Prague, Czech Republic
laroslav Vecer Institute of Physics Charles University Prague, Czech Republic
Vladimir Vondrejs Department of Genetics and
Microbiology Faculty of Natural Sciences Charles University Prague, Czech Republic
Edgar Wagner Institute of Biology II, Botany Albert-Ludwigs-University Freiburg im Breisgau, Germany
Georges A. Wagnieres Institute of Environmental Engineering Swiss Federal Institute of Technology Lausanne, Switzerland
Frank Wehner Max-Planck-Institute for Molecular
Physiology Dortmund, Germany
Nick S. White Oxford University Plant Sciences Department Oxford OXI 3RB, UK
Jacek Wierzchowski Department of Physical Chemistry and
Department of Physiology Faculty of Pharmacy, Medical Academy Warsaw, Poland.
Michael H.F. Wilkinson Department of Medical Microbiology University of Groningen Groningen, The Netherlands
1. L. Wood Oxford University Plant Sciences Department Oxford OXI 3RB, UK
Piotr Wroczynski Department of Physical Chemistry and
Department of Physiology Faculty of Pharmacy, Medical Academy Warsaw, Poland
Jun-Rui Yang Covalent Associates, Inc. Woburn, MA 01801, USA
Zuzana Zemanova Third Medical Department General Faculty Hospital Charles University Prague, Czech Republic
xi
PREFACE
Fluorescence microscopy images can be easily integrated into current video and computer image processing systems. People like visual observation; they like to watch a television or computer screen, and fluorescence techniques are thus becoming more and more popular. Since true in vivo experiments are simple to perform, samples can be directly seen and there is always the possibility of manipulating the samples during the experiments; it is an ideal technique for biology and medicine.
Images are obtained by a classical (now called wide-field) fluorescence microscope, a confocal scanning microscope, upright or inverted, with epifluorescence or transmission. Computerized image processing may improve definition, and remove glare and scattered light signal. It also makes it possible to compute ratio images (ratio imaging both in excitation and in emission) or lifetime imaging. Image analysis programs may supply a great deal of additional data of various types, starting with calculations of the number of fluorescent objects, their shapes, brightness, etc. Fluorescence microscopy data may be complemented by classical measurement in the cuvette yr by flow cytometry.
Fluorescent probes are dyes that are environmentally sensitive. They can be employed as tiny molecular reporters that, encoded in the fluorescence signal, pass on detailed information about their molecular neighborhood. Their location can be determined from fluorescence microscopy images. From measurement of fluorescence intensity, quantum yield, emission spectrum, excitation spectrum, polarization (anisotropy), lifetime, time-resolved fluorescence intensity, and time-resolved fluorescence polarization, one can obtain data on environmental polarity, membrane or cytosol fluidity, membrane potential, and intracellular and intraorganellar ionic composition (pH, calcium, magnesium, sodium, potassium, chloride, iron, zinc, and heavy metals). Furthermore, fluorescent molecules can be used as specific labels to mark any type of cell structure in vivo. There are various fluorescent or fluorogenic enzyme substrates and fluorescent analogues (lipids, nucleosides, lectins). The catalogues list literally hundreds of fluorescent dyes; they are quite alluring.
The immense potential inherent in fluorescent techniques may be illustrated by the example of intracellular ion measurement. In the past, each measurement gave only one single value of ion concentration, while ion-sensitive fluorescent dyes yield maps of intracellular ion concentrations with spatial resolution of 200 nm, millisecond temporal resolution, possibility of 3-D reconstruction, and ion concentration changes as small as 0.0 I pH or 20 nM [Ca2+]. The enormous amount of new data yielded some surprising results, e.g., calcium waves moving across the cell cytoplasm, calcium spikes in individual cells, and various pH and calcium heterogeneities.
The progress in fluorescence instrumentation offers further improvements. Sensitivity can be boosted with single-photon correlation spectroscopy to the observation of single molecules. The definition limit of 150 to 200 nm can be improved by video-enhanced contrast to 20 nm; fluorescent antibody markers can be seen even at a size of 5 to 10 nm.
xiii
Recently developed scanning near-field optical microscopy allows a 50 nm definition for flat biological objects.
The future of fluorescent probes will be probably directed to a simultaneous application of several dyes. Using selective excitation, selective detection, delayed fluorescence dyes, and lifetime imaging, it is easy to monitor several physiological parameters (membrane potential, pH, calcium) simultaneously. I believe that confocal microscopy supplemented with lifetime imaging will become common during the next few years.
The following volume comprises plenary lectures and papers issuing from the conference "FLUORESCENCE MICROSCOPY AND FLUORESCENT PROBES," which took place in Prague from June 25th to 28th, 1995. The selection of well-known scientists for plenary lectures resulted in a surprisingly large number of participants, some of whom learned about the conference at the last moment. The conference presented 90 posters by more than 180 participants from 20 countries. There was an exhibition of fluorescence microscopes supplemented by technical lectures by all leading manufacturers. Plenary lectures, poster sessions, exhibitions, and technical lectures together with a rich social program, created a remarkably exciting atmosphere. This book tries to recall the memories of the conference and to invite the readers to the next Prague conference in April 1997.
Jan Slavik
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CONTENTS
FLUORESCENCE MICROSCOPY AND FLUORESCENT PROBES
Fluorescence Microscopy: State of the Art 0 0 0 000000000000000000000000000000000
B. Herman
Fluorescence Lifetime-Resolved Imaging Microscopy: A General Description of Lifetime-Resolved Imaging Measurements 000000000000000000000000 000 15
Ro Mo Clegg and Po Co Schneider
Confocal Fluorescence Lifetime Imaging 000000000000000000000000000000000 000 35 Ho Co Gerritsen
Multidimensional Fluorescence Microscopy: Optical Distortions in Quantitative Imaging of Biological Specimens 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 47
No So White, R. Jo Errington, Mo Do Fricker, and Jo L. Wood
Fluorescent Probes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 57 Jo Slavik
Flow Cytometry versus Fluorescence Microscopy 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 61 Jo -Eo O'Connor
Multichannel Fluorescence Microscopy and Digital Imaging: On the Exciting Developments in Fluorescence Microscopy 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 67
Ho Gundlach
Fluorescence Lifetime Imaging and Spectroscopy in Photobiology and Photomedicineo 0000000000000000000000000000000000000000000000000 71
Ho Schneckenburger, Mo Ho Gschwend, Ko Konig, Ro Sailer, and W. So L. StrauB
A Versatile Time-Resolved Laser Scanning Confocal Microscope 00000000000000000 79 I. V. Eigenbrot, B. Crystall, and Do Phillips
ION-SENSITIVE FLUORESCENT PROBES
Disappearance of Cytoplasmic Ca2+ Oscillations Is a Sensitive Indicator of Photodamage in Pancreatic J3-Cells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 85
Ao Tengholm and E. Grapengiesser
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Distribution of Individual Cytoplasmic pH Values in a Cell Suspension ............ 91 P. Cimprich and J. Slavik
The Effect of Lysosomal pH on Lactoferrin-Dependent Iron Uptake in Tritrichomonas foetus ............................................ 95
M. Gregor, J. Tachezy, and J. Slavik
On the Protein-Error of the Calcium-Sensitive Fluorescent Indicator Fura-Red ...... 101 N. Opitz, T. Porwol, E. Merten, and H. Acker
Cytoplasmic Ion Imaging: Evidence for Intracellular Calibration Heterogeneities of Ion-Sensitive Fluoroprobes ........................................ 107
N. Opitz, T. Porwol, E. Merten, and H. Acker
The Effect of Protein Binding on the Calibration Curve of the pH Indicator BCECF .. 113 J. Plasek, J. Jaap ter Horst, M. Ameloot, and P. Steels
Artifacts in Fluorescence Ratio Imaging ..................................... 119 P. Cimprich and J. Slavik
Use of Fluorescent Probes and CLSM for pH-Monitoring in the Whole Plant Tissue: pH Changes in the Shoot Apex of Chenopodium rubrum Related to Organogenesis .................................................. 125
J. T. P. Albrechtova, J. Slavik, and E. Wagner
Spatial Resolution of Cortical Cerebral Blood Flow and Brain Intracellular pH as Measured by in Vivo Fluorescence Imaging ........................... 133
R. E. Anderson, F. B. Meyer, and T. M. Sundt, Jr.
MEMBRANE POTENTIAL-SENSITIVE FLUORESCENT PROBES
Is a Potential-Sensitive Probe diS-c3(3) a Nemstian Dye?: Time-Resolved Fluorescence Study with Liposomes as a Model System ................ 139
P. Hetman, J. Vecer, and A. Holoubek
Kinetic Behavior of Potential-Sensitive Fluorescent Redistribution Probes: Modelling of the Time Course of Cell Staining ........................ 145
J. Vecer and P. Herman
Speed of Accumulation of the Membrane Potential Indicator dis-c3(3) in Yeast Cells 151 B. Denksteinova, D. Gaskova, P. Hetman, J. Vecer, K Sigler, J. Plasek, and J.
Malinsky
Spectral Effects of Slow Dye Binding to Cells and Their Role in Membrane Potential Measurements .......................................... 157
1. Plasek and K. Sigler
Exploitation of Rhodamine B in the Killer Toxin Research ...................... 163 L. Valasek, V. Vondrejs, M. Bartunek, and B. Janderova
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FLUORESCENT PROBES FOR NUCLEIC ACIDS
"In Situ" Estimates of the Spatial Resolution for "Practical" Fluorescence Microscopy of Cell Nuclei ........................................ 169
B. Rinke, J. Bradl, B. Schneider, M. Durm, M. Hausmann, H. Ludwig, and C. Cremer
Requirements for a Computer-Based System for FISH Applications ............... 175 W. Malkusch
Fluorescent Dyes and Dye Labelled Probes for Detection of Nucleic Acid Sequences in Biological Material ............................................ 179
I. Durrant and L. Gubba
Fluorescence in Situ Hybridization (FISH) in Cytogenetics of Leukemia ........... 185 K. Michalova, Z. Zemanova, J. Bfezinova, and V. Michalova
Estimation of "Start" in Saccharomyces cerevisiae by Flow Cytometry and Fluorescent Staining of DNA and Cell Protein ........................ 191
L. Guldfeldt, H. Siegumfeldt, L. Lammert, and M. Jakobsen
Fluorescence Image Cytometry of DNA Content: A Comparative Study of Three Fluorochromes and Four Fixation Protocols .......................... 197
M. Benchaib, R. Delorme, P. -A. Bryon, and C. Souchier
FLUORESCENT LABELS, FLUORESCENT AND FLUOROGENIC SUBSTRATES
In Vivo Tissue Characterization Using Environmentally Sensitive Fluorochromes .... 203 G. A. Wagnieres, S. Iinuma, K. T. Schomacker, T. Deutsch, and T. Hasan
Sensitive and Rapid Detection of B-Galactosidase Expression in Intact Cells by Microinjection of Fluorescent Substrate .............................. 211
O. T. Brustugun, G. Mellgren, B. T. Gjertsen, R. Bjerkvig, and S. O. Deskeland
Fluorogenic Substrates Reveal Genetic Differences in Aldehyde-Oxidating Enzyme Patterns in Rat Tissues ........................................... 217
J. Wierzchowski, P. Wroczynski, E. Interewicz, I. Orlanska, and J. Przybylski
Binding of Prothrombin Fragment 1 to Phosphatidylserine Containing Vesicles: A Solvent Relaxation Study ....................................... 223
R. Hutterer and M. Hof
New Thiol Active Fluorophores for Intracellular Thiols and Glutathione Measurement ................................................... 229
M. E. Langmuir, J. -R. Yang, K. A. LeCompte, and R. E. Durand
Quantification of Macro phages in the Cardiovascular System ofHypercholesterolernic Rabbits by Use of Digital Image Processing ........................... 235
J. Metz, P. Pavlov, R. Kinscherf, C. Kohler, and C. Usinger
Fluorescence Assay for Studying P-Glycoprotein Function at Single Cell Level ..... 241 L. Homolya, M. Miiller, Z. Hollo, and B. Sarkadi
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Alterations of Vim entin-Nucleus Interactions as an Early Phase in Cholesterol Oxide-Induced Endothelial Cell Damage ............................. 247
G. Palladini and G. Bellomo
Fluorescence Microscopy of Rye Cell Walls from Kernels to Incubated Doughs ..... 253 M. Koskinen, T. Parkkonen, and K. Autio
Practical Approach for Immunohistochemical Staining of Muscle Biopsies ......... 257 V. Maravic-Stojkovic and V. Baltic
DIGITAL IMAGE ANALYSIS
Rapid Automatic Segmentation of Fluorescent and Phase-Contrast Images of Bacteria 261 M. H. F. Wilkinson
Use of Confocal Microscopy for Absolute Measurement of Cell Volume and Total Cell Surface Area ................................................ 267
C. P. Rubbi and D. Rickwood
Cell Volume Measurements Using Confocal Laser Scanning Microscopy ........... 273 H. Tinel, F. Wehner, and R. K. H. Kinne
Subcellular Cytofluorometry in Confocal Microscopy .......................... 279 D. Demandolx and 1. Davoust
Application of Confocal Microscopy to 3-D Reconstruction and Morphometrical Analysis of Capillaries ........................................... 285
L. Kubinova, M. Jirkovska, P. Hach, D. Palous, P. Karen, and I. Krekule
Retrieving Spatio Temporal Information from Confocal Data: A Study Using Melanotrope Cells of Xenopus laevis ................................ 291
w. J. H. Koopman, B. G. Jenks, E. W. Roubos, and W. J. J. M. Scheenen
Dynamics of Actin Measured by Fluorescence Correlation Microscopy (FCM) ...... 297 B. R. Terry and E. K. Matthews
Index ................................................................. 303
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