This book provides a comprehensive account of the theory of image formation in a confocal fluorescence microscope as well as a practical guideline to the operation of the instrument, its limitations, and the interpretation of confocal microscopy data. The appendices provide a quick reference to optical theory, microscopy-related formulas and definitions, and Fourier theory.
CONTENTS
Symbols and abbreviations
Preface
- 1 Confocal fluorescence microscopy
- 1.1 The principle
- 1.2 The theory
- 1.3 What is the resolution?
- 1.4 Optical aberrations
- 2 Implementation
- 2.1 The microscope objective
- 2.2 Scanning
- 2.3 The detection pinhole
- 2.4 Fluorophores
- 2.4.1 Absorption and fluorescence
- 2.4.2 Fluorescent proteins
- 2.5 The laser
- 3 Practical limits
- 4 Digitisation
- 5 Miscellaneous topics
- 5.1 PSF Measurement
- 5.2 Restoration/deconvolution
- 5.3 The commercial instrument
- 5.4 Related three-dimensional fluorescence techniques
- 5.4.1 4? microscopy
- 5.4.2 Two-photon absorption microscopy
- A Elements of optical theory
- A.1 Geometrical optics
- A.2 Aberration theory
- A.3 Diffraction
- B Formulas, relations and definitions
- C Fourier theory
Bibliography
Index
PREFACE
Since its introduction in the late seventies, the confocal fluorescence microscope has
advanced rapidly from a complex instrument that could be used by specialists only, to a
commercial product, which is part of the standard repertoire of modern biological research.
Its unique feature is that it combines optical techniques?? which are noninvasive, and can
be used inside (intact) watery structures??with high resolution 3D microscopy. In addition,
the use of biochemical-staining techniques provides unprecedented biomolecular specificity.
The contents of this book started out as material for a text that I wrote for the Ph.D.
course Confocal Light Microscopy: Fundamentals and Biological Applications, which was first
given by me in 1996 at the University of Amsterdam. The goal of this course was to
introduce students with a biology background to some of the fundamental concepts of image
formation in confocal fluorescence microscopy and to make them aware of experiment related
issues, such as optical aberrations, bleaching, point spread function measurement and
digitization. Over the years the material for the course has expanded but the goal remained
the same: teaching physics to biologists in such a way that it provides a practical
guideline for the operation of the microscope and the interpretation of microscopic data.
In addition to this, the text was used to compile, over the years, optical formulas and
relations that are used in microscopy related from a multitude of sources. In this way the
book also serves as a quick reference to optical theory in general and its application to
confocal fluorescence microscopy in particular.
The book is organised in two parts. The first five chapters cover the main aspects of
confocal fluorescence microscopy: image formation, practical limitations, fluorescence,
laser operation, and digitization. The appendices provide background material for those who
are not familiar with the basic physics of optics: geometrical optics, diffraction, and
Fourier theory.
I have been very fortunate to start as a post-doc in the group of Prof. G.J. Brakenhoff,
one of the godfathers of confocal microscopy. His support and expertise have been
invaluable. I would also like to express my appreciation to the department of Molecular
Cytology of the Swammerdam Institute for Life Sciences at the University of Amsterdam for
giving me the opportunity to teach the confocal microscopy course to Ph.D. students. Over
the years a number of colleagues have assisted me by reviewing the manuscript. In this
regard I would like specifically to thank Arjan Buist, Rick Ghauharali and Sjors Wurpel. I
would also like to thank Wijnand Takkenberg for technical assistance with the confocal
microscope.
Michiel Mueller
Amsterdam, June 2005
