Table Of ContentFrom Cells to Proteins:
Imaging Nature across Dimensions
NATO Security through Science Series
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Series B:Physics and Biophysics – Vol.3
From Cells to Proteins:
Imaging Nature across Dimensions
edited by
Valtere Evangelista
CNR Institute of Biophysics, Pisa, Italy
Laura Barsanti
CNR Institute of Biophysics, Pisa, Italy
Vincenzo Passarelli
CNR Institute of Biophysics, Pisa, Italy
and
Paolo Gualtieri
CNR Institute of Biophysics, Pisa, Italy
Published in cooperation with NATO Public Diplomacy Division
Proceedings of the NATO Advanced Study Institute on
From Cells to Proteins:Imaging Nature across Dimensions
Pisa, Italy
12-23 September 2004
A C.I.P.Catalogue record for this book is available from the Library of Congress.
ISBN-10 1-4020-3615-9 (PB)
ISBN-13 978-1-4020-3615-6 (PB)
ISBN-10 1-4020-3614-0 (HB)
ISBN-13 978-1-4020-3614-9 (HB)
ISBN-10 1-4020-3616-7 (e-book)
ISBN-13 978-1-4020-3616-3 (e-book)
Published by Springer,
P.O.Box 17, 3300 AADordrecht, The Netherlands.
www.springeronline.com
Printed on acid-free paper
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© 2005 Springer
No part of this work may be reproduced, stored in a retrieval system, or transmitted in
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Printed in the Netherlands.
In memory of Antonietta Barsanti
v
CONTENTS
Preface........................................................................................................xi
Optical Microscopy for Cell Imaging..........................................................1
J. M. Girkin
Introduction to Transmission and Scanning Electron Microscopy............23
F. Verni and S. Gabrielli
Potential and Limitation of Cytochemistry................................................37
G. Rosati and S. Gabrielli
Cryotechniques for Electron Microscopy: A Minireview..........................53
P. Lupetti
Cell Membrane Specializations as Revealed by the Freeze-Fracture
Technique..............................................................................71
R. Dallai
Imaging, Measuring and Manipulating Biological Matter from the
Millimeter to Nanometer Scale.........................................85
D. Stoffler and U. Aebi
Inside the Small Length and Energy Scales of the World of the
Individual Biological Molecule...........................................111
M. Sandal, G. Zuccheri and B. Samorì
Scanning Probe Microscopy across Dimensions.....................................139
C. Gebeshuber, R. A. P. Smith, H. Winter and F. Aumayr
Imaging Cells Using Soft X-Rays...........................................................167
T. W.Ford
From Microscopy to Nanoscopy: How to Get and Read Optical
Data at Single Molecule Level Using Confocal and
Two-Photon Excitation Microscopy...................................187
A. Diaspro, P. Bianchini, V. Caorsi, D. Mazza, M. Pesce,
I. Testa, G. Vicidomini, G. Chirico, F. Cannone and C. Usai
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viii
Fluorescence Resonance Energy Transfer (FRET) and Fluorescence
LifetimeImaging Microscopy (FLIM .............................209
T. M. Jovin, D. S. Lidke and E. A. Jares-Erijman
Quantum Dots, a New Tool for Real-time in Vivo Imaging....................217
D. J. Arndt-Jovin, T. M. Jovin and D. S. Lidke
Image Acquisition and its Automation in Fluorescence Microscopy......227
M. Kozubek
Stereological and Digital Methods for Estimating Geometrical
Characteristics of Biological Structures Using Confocal
Microscopy..........................................................................271
L. Kubínová, J. Janá(cid:254)ek, J. Albrechtová and P. Karen
Wavelength as the Fourth Dimension in Light Microscopy....................323
V. Evangelista, L. Barsanti, A.M. Frassanito, V. Passarelli
and P. Gualtieri
Time as the Fifth Dimension in Microscopy...........................................335
P. Coltelli, L. Barsanti, V. Evangelista, A. M. Frassanito and
P.Gualtieri
Image Deconvolution...............................................................................349
M. Bertero and P. Boccacci
Image Formation in Fluorescence Microscopy: Three-Dimensional
Mathematical Model...........................................................371
G. Vicidomini
Atomic Force Microscopy Study on the Pellicle of the Alga
Euglena Gracilis..................................................................395
F. Sbrana, L. Barsanti, V. Passarelli and P. Gualtieri
Atomic Force Microscopy Study of Pili in the Cyanobacterium
Synechocystis Sp. Pcc 6803.................................................405
E. V. Dubrovin, I. A. Kirik, M. M. Babykin and I. V. Yaminsky
ix
Changes of Algae Protein Complex under Ph Effect...............................415
T.V. Parshikova
Leaf Fluorescence as Diagnostic Tool for Monitoring Vegetation..........423
S. Apostol
Dynamic Holography for Study of Nonlinear Optical Processes in
Biological Photoreceptor Molecule.....................................431
E. Korchemskaya, D. Stepanchikov, S. Bugaychuk and
N. Burykin
“Seeing" Lipid Membranes by Solid-State NMR....................................451
V. Castro
Visuomotor Coordination in Behaviour of Pigeons Following Post-
hatching Monocular Experience: An Image Analysis
Study...................................................................................459
G. Cesaretti, C. Kusmic and D. Musumeci
Index........................................................................................................471
List of Contributors..................................................................................475
PREFACE
This volume contains the lectures and seminars given at the NATO
Advanced Study Institute on “From cell to proteins: Imaging nature across
dimensions”, held in Pisa, Italy in September, 2004. The Institute was
sponsored and mainly funded by the Scientific Affairs Division of NATO.
The European Biophysical Societies’ Association also supported the School
and contributed to its success. It is my pleasant duty to thank these
institutions. This ASI offered updated information on how many information
we are able to achieve in the exploration of the inner details of biological
specimens in their native structure and composition.
How deep we can see inside Nature smallest secrets? Will it be possible
someday in a near future to investigate living structures at atomic level? This
area of study is very interdisciplinary, since it uses and it is based on the
principles and techniques of biology, physics, chemistry, mathematics, and
engineering to elucidate the structures of biological macromolecules, of
supramolecular structures, organelles, and cells. Scientists from very
different backgrounds contributed to the understanding of this fast
developing field of research, which has seen considerable progress during
the last years. These revelations came not (only) through biochemistry and
molecular biology, but through staggering improvements of microscopy.
The widest range of dimensions is covered by the electron microscope
(EM): it can image whole cells, their organelles, cytoskeleton and
supramolecular assemblies, as well as individual biomolecules, their
submolecular structure and, ultimately, individual atoms. Recently, the
scanning force microscope (SFM) has opened completely new perspectives
for analyzing the surface topography of biological matter in its aqueous
environment at a resolution comparable to that achieved by EM. Most
exciting, the SFM is the first imaging device allowing direct correlation
between structural and functional states of biomolecules at submolecular
resolution. The developments of implementation of laser beam and stage
scanning systems incorporating confocal optics, the advent of new electro-
optical detectors with great sensitivity, linearity, and dynamic range, the
possibility of 2D fast image enhancement, reconstruction, analysis and 3D
display, and application of luminescence techniques gives the possibility to
investigate the chemical and molecular details of life processes, and we can
say that this digital approach, comprehending FLIMT and FRET techniques
combined with the use of quantum dots, can probe the spatio-temporal
organization of cell metabolism. Moreover, through the use of antibody
labelling and confocal or multiphoton microscopy joined to algorithms for
image restoration, it is now commonplace to distinguish and study structures
an order of magnitude below the theoretical limit of resolution of the light
microscope (about 250 nm). Also, individual 20 nm particles carrying
xi
xii
specific protein probes can be seen, for example tracking across the surface
of a living cell.
We can conclude that with the microscope we can explore the
microcosm, from tissues to cells and all the way down to molecules and
atoms, and that this ASI represented a successful opportunity for carrying on
and implementing an interdisciplinary approach to the study of theoretical
and practical aspects of this research field.
The first part of the book deals with a general overview of the state of the
art of microscopy including theoretical aspects, whereas the second part
deals with uses and applications of the different kind of microscopes.
I do hope this book has caught the spirit in which the ASI was conceived.
Paolo Gualtieri
Director of the ASI “From cell to proteins: Imaging nature across
dimensions”
