Introduction to Confocal Fluorescence Microscopy, Second Edition

Author(s): Michiel Mueller

Published: 2005

PDF ISBN: 9780819481177 | Print ISBN: 9780819460431

DESCRIPTION

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.
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Front Matter

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CHAPTER 1.

Confocal Fluorescence Microscopy

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1.1. The principle
1.2. The theory
1.2.1. The point spread function (PSF)
1.2.2. Validity of approximations
1.2.3. The PSF in 3D
1.2.4. Image formation
1.2.5. The confocal PSF
1.2.6. The optical transfer function (OTF)
1.3. What is the resolution?
1.3.1. Resolution and the OTF
1.3.2. Magnification
1.4. Optical aberrations
1.4.1. Chromatic aberration
1.4.2. Refractive index mismatch

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CHAPTER 2.

Implementation

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2.1. The microscope objective
2.1.1. Magnification, NA and immersion
2.1.2. Tube length
2.1.3. Cover glass
2.1.4. Microscope objective types
2.1.5. Working distance
2.1.6. Transmission
2.2. Scanning
2.2.1. Specimen scanning
2.2.2. Laser scanning
2.2.3. The Nipkow disk
2.2.4. Bilateral scanning
2.3. The detection pinhole
2.4. Fluorophores
2.4.1. Absorption and fluorescence
2.4.2. Molecular energy levels
2.4.3. Absorption
2.4.4. Vibrational and rotational relaxation
2.4.5. Emission
2.4.6. Phosphorescence
2.4.7. Bleaching
2.4.8. Fluorescent proteins
2.5. The laser

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CHAPTER 3.

Practical Limits

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CHAPTER 4.

Digitization

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4.1. Discrete sampling of continuous signals
4.2. Digitization in the spatial domain
4.3. Digitization in the intensity domain

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CHAPTER 5.

Miscellaneous Topics

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5.1. PSF measurement
5.1.1. Preparation protocol
5.2. Restoration/deconvolution
5.2.1. The principle
5.2.2. Practical considerations
5.2.3. Practical example
5.3. The commercial instrument
5.3.1. Excitation
5.3.2. Objectives
5.3.3. Dichroics
5.3.4. Detection pinhole
5.3.5. Detection
5.3.6. Speed
5.3.7. Software
5.4. Related 3D fluorescence techniques
5.4.1. 4π-microscopy
5.4.2. Two-photon absorption microscopy

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Appendix A Elements of Optical Theory

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A.1. Geometrical optics
A.1.1. Imaging and magnification
A.2. Aberration theory
A.2.1. The primary aberrations
A.2.2. Chromatic aberrations
A.2.3. Zernike polynomials

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Appendix B Formulae, Relations, and Definitions

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B.1. Optical coordinates
B.2. Focal field distribution
B.3. Classical theory versus full diffraction theory
B.4. Confocal PSF
B.4.1. Elongation of the PSF
B.4.2. Cut-off frequency of the OTF
B.4.3. Microscope objective

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Appendix C Fourier Theory

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C.1. Properties of the Fourier transform

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Back Matter

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