Spie Press BookIntroduction to Confocal Fluorescence Microscopy, Second Edition
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Symbols and abbreviations
- 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
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.
Amsterdam, June 2005