Imaging of Nucleic Acids and Quantitation in Photonic Microscopy

181 Pages·2001·4.96 MB·English

by  Xavier Ronot

IMAGING of
NUCLEIC ACIDS
and QUANTITATION
in PHOTONIC
MICROSCOPY
Methods in Visualization
Series Editor: Gérard Morel
In Situ Hybridization in Light Microscopy
Gérard Morel and Annie Cavalier
Visualization of Receptors: In Situ Applications
of Radioligand Binding
Emmanuel Moyse and Slavica M. Krantic
Genome Visualization by Classic Methods
in Light Microscopy
Jean-Marie Exbrayat
Imaging of Nucleic Acids and Quantitation
in Photonic Microscopy
Xavier Ronot and Yves Usson
In Situ Hybridization in Electron Microscopy
Gérard Morel, Annie Cavalier,
and Lynda Williams
IMAGING of
NUCLEIC ACIDS
and QUANTITATION
in PHOTONIC
MICROSCOPY
Edited by
Xavier Ronot
Professor at the Ecole Pratique des Hautes Etudes
Yves Usson
Senior Researcher at the
Centre National de la Recherche Scientifique
CRC Press
Boca Raton London New York Washington, D.C.
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Cover inset: Confocal 3-D reconstruction of HeLa cell mitotic nuclei after heat shock treatment. chromosomes
are shown in red, and HSF1 transcription factors are shown in green. For details, see Jolly, Usson, and
Morimoto, PNAS, 1998, 96: 6769–6774.
Library of Congress Cataloging-in-Publication Data
Imaging of nucleic acids and quantitation in photonic microscopy / edited by Yves Usson,
Xavier Ronot.
p. cm. — (Methods in visualization)
ISBN 0-8493-0817-8 (alk. paper)
1. Nucleic acids—Analysis. 2. Fluorescent probes. 3. Fluorescence microscopy. I.
Usson, Yves. II. Ronot, Xavier. III. Series.
QP620 .I43 2001
572.8'028—dc21 00-140132
CIP
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Library of Congress Card Number 00-140132
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SERIES PREFACE
Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge for
cell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecular
biology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizing
methods (photonic and electronic microscopy), but also for more recent methods (confocal and
scanning tunneling microscopy). These visualization techniques have gained so much in specificity
and sensitivity that many researchers are considering expansion from in-tube to in situ experiments.
The application potentials are expanding not only in pathology applications but also in more-
restricted applications such as tridimensional structural analysis or functional genomics.
This series addresses the need for information in this field by presenting theoretical and technical
information on a wide variety of related subjects: in situ techniques, visualization of structures,
localization and interaction of molecules, functional dynamism in vitro or in vivo.
The tasks involved in developing these methods often deter researchers and students from using
them. To overcome this, the techniques are presented with supporting materials such as governing
principles, sample preparation, data analysis, and carefully selected protocols. Additionally, at every
step we insert guidelines, comments, and pointers on ways to increase sensitivity and specificity,
as well as to reduce background noise. Consistent throughout this series is an original two-column
presentation with conceptual schematics, synthesizing tables, and useful comments that help the
user to quickly locate protocols and identify limits of specific protocols within the parameter being
investigated.
The titles in this series are written by experts who provide to both newcomers and seasoned
researchers a theoretical and practical approach to cellular biology and empower them with tools
to develop or optimize protocols and to visualize their results. The series is useful to the experienced
histologist as well as to the student confronting identification or analytical expression problems. It
provides technical clues that could only be available through long-time research experience.
Gérard Morel, Ph.D.
Series Editor
V
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PREFACE
The discovery of the cell nucleus in 1831 was the first step in the story of cell and nucleic acid
analysis. The cell nucleus governs the biosynthetic events that characterize the cell’s past and
future. At the molecular level, the cell nucleus is designed to produce, or direct the production of,
molecules essential for cell life and necessary for cell proliferation, differentiation, and death. This
activity is programmed by key elements: the nucleotide sequences in the deoxyribonucleic acid
of the chromatin.
Researchers interested in nuclear structure and function feel the need to establish relationships
between morphology and function and activity, and to search for cytological clues to regulatory
and control mechanisms. Visualization of nuclear components has introduced new dimensions to
the biology of the nucleus and new insights into its chemistry.
Nuclear organization studies follow two main lines: biochemical analysis of isolated nuclei and
cytological analysis in living or fixed cells. The second approach probes and explores nuclear
components inside the cell using microscopy.
Imaging of Nucleic Acids and Quantitation in Photonic Microscopy was undertaken to serve as
a tutorial for all researchers interested in the analysis of nucleic acids by image cytometry following
fluorescent labeling and detection procedures.
This book belongs to the series entitled Methods in Visualization. Its special feature is to cover
theoretical bases and to highlight the critical points, pro and cons, dos and don’ts resulting from
the authors’ experience. Chapters are written by researchers with extensive experience in the use
of photonic microscopy in the field of nucleic acid research.
The design of probes that enable direct or indirect labeling of nucleic acids is discussed in
Chapters 1 and 2. Other chapters describe methodologies developed for microscopic analysis of
nucleic acids: single cell electrophoresis (comet assay) to detect DNA lesions (Chapter 3), and
fluorescent in situ hybridization of artificially decondensed DNA (Chapter 4). Chapters 5, 6, and
7 describe fluorescent imaging principles for the in situ study of nuclear distribution and colocal-
ization of molecules (epi-illumination, confocal, two-photon microscopes, and associated captors),
different steps in image processing in the case of fluorescently labeled objects. Chapter 8 discusses
examples of quantification: chromatin organization in cells maintained alive and distribution of
nucleolar bodies inside the nucleus (AgNOR).
The amount of information provided by photonic microscopy contributes widely to the under-
standing of nuclear function and related components.
VII
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ACKNOWLEDGMENTS
This book was written in the framework of the European “Leonardo da Vinci” project (Grant
F/96/2/0958/PI/II.I.1c/FPC), in association with Claude Bernard-Lyon 1 University (http://
brise.ujf-grenoble.fr/LEONARDO).
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