Modern Optics, Electronics and High Precision Techniques in Cell Biology

252 Pages·1998·9.55 MB·English

by  Cora-Ann Schoenenberger

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Springer
Berlin
Heidelberg
New York
Barcelona
Budapest
Hong Kong
London
Milan
Paris
Santa Clara
Singapore
Tokyo
G. Isenberg (Ed.)
Modern Optics,
Electronics
and High Precision
Techniques
'in Cell Biology
With 120 Figures, 13 in Color
Springer
PROFESSOR
DR. DR. GERHARD ISENBERG
Technical University of Munich
Dept. of Biophysics E-22
Ja mes-Franck- Strasse
85747 Garching/Munich
Germany
ISBN-13:97S-3-642-S0372-7
CIP Data applied for
Die Deutsche Bibliothek - CIP-Einheitsaufnahme
Modern optics, electronics and high precision techniques in cell biology I Gerhard Isenberg (ed.).
- Berlin; Heidelberg; New York; Santa Clara; Singapore; Tokyo: Springer, 1997
ISBN-13:978-3-642-80372-7 e-ISBN-13:978-3-642-80370-3
DOl: 10.1007/978-3-642-80370-3 DBN 95.083379.7
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Preface
In spite of tremendous scientific progress over the past years, cell biologists
do not yet understand the fundamental processes that determine the life cy
cle of a cell. Such are: cell movement and cell spreading, cell division, cell
communication, cell signaling, cell regeneration and cell death. Biochemistry
has enabled us to recognize and to isolate an overwhelming number of new
proteins. In vitro assays and the reinjection of proteins into cells and tissues
have provided insights into molecular functions and cellular mechanisms.
The renaissance of the genetic approach by applying restriction enzymes and
vectors, PCR and antisense technology has enabled us to overexpress certain
cellular products, to make altered constructs of cell components or to create
"knock-out" mutants that entirely lack the factor of interest. Amazingly en
ough, all these molecular toys have led to a stream of information but not, in
a comparable degree, to a better understanding. Has the puzzle become too
complex to get solved; or are the windows too small that we are looking
through?
As an attempt to answer both questions, the aim of the present mono
graph Modern Optics, Electronics and High Precision Techniques in Cell Biol
ogy is first to provide cell and molecular biologists with a whole new scope
of easily applicable techniques including brand-new optical, biophysical,
physicochemical and biosensoric devices. Secondly, these newly developed
techniques allow us to look at cells and biological systems as a whole. This
means that cells and molecular systems can be analyzed in real life, i.e. at a
real place, in real time and in real dimensions.
The optical section begins with a description of the principles and appli
cation of atomic force microscopy (AFM) by Drs. C.-A. Schoenenberger,
D. J. Muller and A. Engel. Imaging of native biological structures, culminat
ing in monitoring and changing molecular conformations of individual mole
cules, is the main topic in this "state-of-the-art" article. Three-dimensional
imaging by optical sectioning through a cell is possible by the newly devel
oped Confocal Laser Scanning Microscopy at the light microscope level (Drs.
Borlinghaus and Grobler).
Cellular neurobiology contributes with two revolutionary novel techniques
to this volume: Drs. Dodt and Zieglgansberger have developed an infrared il
lumination which, in combination with a gradient contrast system and video
enhancement, allows monitoring of development and plasticity of neuronal
networks in their natural environment.
Exciting time-resolved imaging of membrane potentials and ion fluxes at
the cellular level may be obtained by a new fiber array photo diode camera,
VI Preface
developed and described by Drs. Hosoi, Tsuchiya, Takahashi, Kashiwasake
Jibu, Sakatani and Hayakawa.
Both approaches open up quite new dimensions for analyzing molecular
nerve cell functions.
Drs. Schliwa and Kas with their collaborators look at individual biopoly
mers with different techniques: Optical Laser Tweezers allow the manipula
tion of single microtubules in order to measure their rigidity and stiffness.
Polymer dynamics of semi-flexible actin rods and a corresponding theory
are derived from high resolution single protein imaging by fluorescence.
Neutron reflection and its application for investigating protein-lipid inter
actions in cell membranes are covered in the chapter by Drs. Bayerl and
Maierhofer. This non-invasive technique is dependent on large nuclear reac
tor facilities and therefore is of increasing interest worldwide. Utilization of
physical techniques for cell science results from the attempt to find a com
mon scientific language among biophysicists and biologists. An example may
also be the following chapter by Drs. Goldmann, Guttenberg, Ezzel and Isen
berg, introducing the stopped-flow technique for measuring fast kinetics of
biological reactions.
Primarily derived from a physical technique, namely, surface plasmon res
onance, is the BIA-CORE system, a biosensor-based analytical technology for
monitoring interactions in real time without using any labeling (Drs. Markey
and Schindler). Both techniques have a wide application range and fill a gap
within the spectrum of cell biological methods.
An intriguing new way of producing micromachined substrates is illus
trated by Drs. Galbraith and Sheetz. Micromachining combined with two-di
mensional micro-electronic sensors gives new insights into the signal proces
sing of neuronal networks. Here, the device is used to record traction forces
of individual cell contacts.
A final example that the new age of cell science significantly relates to bio
engineering is given by Dr. Sackmann, who describes the operation of high
precision viscometers designed to measure the viscoelasticity of biological
polymers.
Each of the new technologies presented here is directly correlated with an
actual field of cell biological research. One can envisage for the next decades
that cell biological research will be determined not only by genetics but will
necessarily have to include modern optics, electronics and high precision
techniques to open up the new era of Molecular Cell Physiology. I dare to
predict that knowledge and understanding are guaranteed when one is open
minded towards new approaches.
Munich, January 1997 G. Isenberg
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
Chapter 1
Atomic Force Microscopy Provides Molecular Details of Cell Surfaces
Cora-Ann Schoenenberger, Daniel J. Muller and Andreas Engel
Chapter 2
Basic Principles and Applications of Confocal Laser Scanning Microscopy 33
Rolf Borlinghaus and Bernhard Grobler
Chapter 3
Visualization of Neuronal Form and Function in Brain Slices
by Infrared Video microscopy ................................ 55
Hans-Ulrich Dodt and Walter Zieglgansberger
Chapter 4
Time-Resolved Imaging of Membrane Potentials and Cytoplasmic Ions
at the Cellular Level with a 50x50 Fiber Array Photo diode Camera .. .. 75
S. Hosoi, H. Tsuchiya, M. Takahashi, M. Kashiwasake-Jibu, K. Sakatami
and T. Hayakawa
Chapter 5
Micromanipulation of Macromolecules: How to Measure the Stiffness
of Single Microtubules ..................................... 89
Harald Felgner, Rainer Frank and Manfred Schliwa
Chapter 6
Dynamics of Single Protein Polymers Visualized
by Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1
Josef Kas, Jochen Guck and David Humphrey
Chapter 7
The Interaction of Proteins with Membrane Surfaces
at Molecular Resolution: The Neutron Reflection Method ........... 139
Thomas M. Bayerl and Andreas P. Maierhofer
VIII Contents
Chapter 8
The Study of Fast Reactions by the Stopped-Flow Method. . . . . . . . . .. 159
Wolfgang H. Goldmann, Zeno Guttenberg, Robert M. Ezzel
and Gerhard Isenberg
Chapter 9
Biomolecular Interactions Analysis (BIA Technology).
A Universal Biosensor-Based Technology for Biochemical Research
and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173
Francis Markey and Franz Schindler
Chapter 10
Measuring Cellular Locomotion Forces with Micro-Machined Substrates 195
Catherine G. Galbraith and Michael P. Sheetz
Chapter 11
Viscoelasticity, Rheology and Molecular Conformational Dynamics
of Entangled and Cross-Linked Actin Networks .................. 211
Erich Sackmann
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 259
CHAPTER 1
Atomic Force Microscopy Provides Molecular
Details of Cell Surfaces
Cora-Ann Schoenenbergerl • Daniel J. Miillerl,2 • Andreas Engell *
Contents
1.1 Introduction 3
1.2 Principles of Atomic Force Microscopy ................. . 4
1.3 Sample Immobilization for Imaging
in Physiological Environment ........................ . 5
1.3.1 Supports ........................................ . 6
1.3.2 Sample Immobilization ............................. . 6
1.4 Imaging of Biomolecular Assemblies ................... . 9
1.4.1 Living Cells ..................................... . 9
1.4.2 Surface and Subsurface of Bovine Articular Cartilage ...... . 10
1.4.3 Intermediate Filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.4.4 Nuclear Pore Complex ............................. . 14
1.4.5 Bacteriophage T4 Polyheads ......................... . 14
1.4.6 E. coli OmpF Porin Surfaces ......................... . 16
1.4.7 Bacteriorhodopsin ................................ . 17
1.4.8 Hexagonally Packed Intermediate Layer ................ . 21
1.5 Identification of Structures Observed by AFM ........... . 21
1.6 Conformational Changes Monitored by AFM ............ . 24
1.7 Conclusions and Perspectives ........................ . 27
References ....................................... 28
1 M.E. Muller Institute for Microscopy, Biozentrum,
University of Basel, CH-4056 Basel, Switzerland
2 IBI-2:Structural Biology, Forschungszentrum Jiilich, D-52425 Jiilich,
Germany
* Corresponding address: M.E. Muller Institute, Biozentrum,
University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland,
Tel.: [phone], Fax: [phone], e-mail: [email protected]
Atomic Force Microscopy Provides Molecular Details of Cell Surfaces 3
1.1
Introduction
Ten years ago, Binnig, Quate, and Gerber presented the microscopy world
with the atomic force microscope (AFM; Binnig et al. 1986). As a member of
the scanning probe microscopes, the AFM provides detailed maps of surface
topographies by raster scanning a fine tip over the sample surface and re
cording the changes in tip-sample interactions. Initially, the AFM found its
applications in material sciences, for example, in the imaging of magnetic
fields above thin-film recording heads (Martin and Wickramasinghe 1987) or
of polymers (Albrecht et al. 1988). It took as little as one year to achieve
atomic resolution on hard flat surfaces (Binnig et al. 1987). Unlike its ances
tor, the scanning tunneling microscope (STM), the AFM does not require the
specimen to be either electron or ion conductive. In addition, it is opera
tional in liquid as well as in air or in vacuo. The possibility of imaging non
conductive samples in aqueous solutions has launched this technology into
biological research. With the AFM, the imaging of native biological struc
tures in a physiological environment at molecular resolution had moved into
the realm of reality. That biological compounds could indeed be imaged in
aqueous solutions was first reported by Drake and colleagues in 1989 who
showed images of rehydrated polyalanine adsorbed to glass and of fibrin
polymers adsorbed to mica in phosphate-buffered saline (Drake et al. 1989).
Compared to these early images, the resolution obtained by AFM has come a
long way in a very short time. A number of technical developements were vi
tal for improving resolution. The optical lever significantly increased the sen
sitivity of the detection of the deflecting cantilever (Meyer and Amer 1988).
Together with the micro fabrication of cantilevers with integrated tips that
have a defined geometry and high-aspect ratios, a more reproducible opera
tion at forces as low as a few hundred picoNewtons was achieved. Further on
in this chapter, we will present examples from our own work where sub mole
cular details of native membrane proteins are resolved. In its infancy, the re
solution obtained with biomolecules was limited, but the potential of AFM to
monitor dynamic biological processes was already recognized early on. The
recent observation of reversible conformational changes of bacteriorhodopsin
(Muller et al. 1995b) and of single pores of the hexagonally packed inter
mediate (HPI) layer (Muller et al. 1996b) have spurred the hope that struc
ture-function relationships can be directly monitored with the AFM.
In this review we will briefly introduce the principle of AFM and then dis
cuss the most important aspect of sample preparation which is its immobili
zation. With respect to the AFM applications in biology, space limitations
oblige us to focus on the imaging of biomolecular assemblies and the struc
tural information contained in these images. We will do so by illustrating the
different degrees of resolution obtained with examples from our own re
search. Other exciting applications, such as the measurement of intermolecu
lar forces (Florin et al. 1994; Lee et al. 1994; Moy et al. 1994; Dammer et al.
1995, 1996) and the measurement of visco-elastic cell surface properties
(Weisenhorn et al. 1993; Hoh and Schoenenberger 1994; Radmacher et al.
1994) merit being discussed independently.