Principle of Fluorescence Microscopy

J.R. Lakowicz Principles of Fluorescence Microscopy (Plenum, New York 1983)... 

This article gives an overview of the current applications of the technique of fluorescence microscopy. The four sections describe, respectively, (1) the basic principles of fluorescence microscopy, (2) the types of information which can be obtained by fluorescence microscopy, (3) the technical ways in which fluorescence microscopy can be adapted to study various chemical species and (4) some examples of the range of biological, mineralogical and artificial specimens that can be studied. 

See, for example. J. R. l,akowicz. Principles of Fluorescence Spectroscopy, 2nd ed.. New York Kluwer Academic Publishing/Plenum Press, 1999 B. Herman, Fluorescence Microscopy. 2nd ed.. New York Springcr-Verlag, 1998. 

By extending the principles of confocal microscopy [71], it became clear that it is possible to collect interference-free Raman maps of small samples in matrices, even if these matrices are strong Raman scatterers or fluorescers. 

Principle A confocal microscopy as a modification of luminescent microscope may produce images of high quality from fluorescing cells and permits the study of cells structures (see Chapter 8). 

As shown in Section 11.2.1.1, more details can be obtained by confocal fluorescence microscopy than by conventional fluorescence microscopy. In principle, the extension of conventional FLIM to confocal FLIM using either time- or frequency-domain methods is possible. However, the time-domain method based on singlephoton timing requires expensive lasers with high repetition rates to acquire an image in a reasonable time, because each pixel requires many photon events to generate a decay curve. In contrast, the frequency-domain method using an inexpensive CW laser coupled with an acoustooptic modulator is well suited to confocal FLIM. 

Bacallao, R., Morgane, B., Stelzer, E. H. K., and DeMey, J. (1989) Guiding principles of specimen preservation for confocal fluorescence microscopy, in Handbook of Biological Confocal Microscopy (Pawley, J. B., ed.). Plenum, New York, pp. 197-205. 

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