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Microscopy Reference Library

Fluorescence Microscopy

The application of fluorescence illumination and detection in optical microscopy has ushered in a wide range of advanced applications for live-cell imaging and in vivo observations. The articles tabulated in this section discuss the basic aspects of fluorescence, microscope configuration, fluorescent probes, software, light sources, detectors, objectives, filter sets, and a variety of other pertinent topics.

Lichtman, J. W. and Conchello, J. A.

Fluorescence Microscopy.  Nature Methods 2: 910-919 (2005).  Highly recommended, this review article covers all of the important aspects necessary for beginners to understand the basic principles underlying fluorescence microscopy. Included are the principles of fluorescence, the characteristics of fluorophores, excitation and emission spectral properties, microscope parameters, light sources, objective lenses, and photobleaching artifacts.

Michalet, X., Kapanidis, A. N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M. and Weiss, S.

The power and prospects of fluorescence microscopies and spectroscopies.  Annual Review of Biophysics and Biomolecular Structure 32: 161-182 (2003).  A comprehensive treatise that discusses basic aspects of fluorescence microscopy and highlights improvements that have enabled imaging in living cells and animals. Fundamentals of fluorescence and fluorophores are described, as are imaging modalities, advanced techniques, and single molecule detection.

Petty, H. R.

Fluorescence microscopy: established and emerging methods, experimental strategies, and application in immunology.  Microscopy Research and Technique 70 687-709 (2007).   Although titled as specialized to immunology, this review article covers a wide spectrum of topics in general fluorescence microscopy that are of interest in many disciplines. Addressed are basic microscope configuration, filter sets, detectors, noise, objectives, resolution, specimen preparation, advanced techniques, and photobleaching.

Stelzer, E. H. K.

Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy.  Journal of Microscopy 189: 15-24 (1998).  This review article addresses several of the most important aspects of fluorescence imaging. The author discusses the point spread function, how contrast and resolution are related, and the effects of pixelation and noise on contrast. In addition, a nice discussion is presented of dynamic range and its relationship with gray levels.

Haustein, E. and Schwille, P.

Trends in fluorescence imaging and related techniques to unravel biological information.  HFSP Journal 1: 169-180 (2007).  In reviewing the current state-of-the art in fluorescence microscopy, the authors briefly discuss introductory concepts before embarking on advanced techniques. Included are explanations and references on confocal, multiphoton, oblique illumination, 4Pi, stimulated emission, structured illumination, FRET, TIRF, FCS, and lifetime imaging.

Wouters, F. S.

The physics and biology of fluorescence microscopy in the life sciences.  Contemporary Physics 47 239-255 (2006).  This review is a discussion of fluorescence microscopy targeted at physicists who have had little experience with biology. Topics include the availability of affordable CCDs and solid state lasers as the result of demands from astronomy, optical communications, and data storage applications, as well as the rapid rise in the number of publications dealing with fluorescent proteins and microscopy.

Brown, C. M.

Fluorescence microscopy: avoiding the pitfalls.  Journal of Cell Science 120: 1703-1705 (2007).  A very readable discourse of potential trouble spots when conducting fluorescence microscopy investigations. Among the topics discussed are bleed-through, fluorophore saturation, undersampling and oversampling, detector saturation, and software settings.

Coling, D. and Kachar, B.

Principles and Application of Fluorescence Microscopy.  Current Protocols in Molecular Biology 14.10.1-14.10.44 (2003).  The authors present a general overview of fluorescence microscopy targeted at students and investigators new to the field. Included are practical discussions of fluorescent probes, filters, light sources, objectives, image resolution, the point spread function, and immunofluorescence.

Wolf, D. E.

Fundamentals of fluorescence and fluorescence microscopy.  Methods in Cell Biology 81: 63-91 (2007).  A review of the principle concepts in fluorescence, including atomic and molecular phenomena, Beer's Law, excited state lifetimes, saturation, decay mechanisms, energy transfer, depolarization, and measuring fluorescence in the steady state. Also included is a discussion of detectors, filters, and an overview of the basic configuration of a fluorescence microscope.

North, A. J.

Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.  Journal of Cell Biology 172: 9-18 (2006).  Although targeted at imaging problems in all forms of optical microscopy, this guide is particularly well suited to address capturing images with fluorescence illumination. The author discusses specimen preparation, mounting media, objective selection, fluorophores, filter sets, aberrations, acquisition, and preparation of images for publication.