fluorescence microscopy and fluorescent probes978-1-4899-1866-6/1.pdf · dortmund, germany lolana...

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,


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


Blanka Janderovci Department of Genetics and


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


Ralf Kinscherf Department of Anatomy and Cell

Biology III University of Heidelberg Heidelberg, Germany

Christoph Kohler Department of Anatomy and Cell


viii

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


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,


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


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


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


Jacek Przybylski Department ofphysical Chemistry and


ix

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,


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

x

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


Claus Usinger Department of Anatomy and Cell Biology III University of Heidelberg Heidelberg, Germany

L.Valasek Department of Genetics and


laroslav Vecer Institute of Physics Charles University Prague, Czech Republic

Vladimir Vondrejs Department of Genetics and


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


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


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

xvii

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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