Imaging Membrane Organization and Dynamics
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In this chapter, we will discuss how the conventional techniques of nonradiative fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) can be extended, by coupling these techniques to low-light-level video microscopy and digital image processing, to provide an additional dimension of information. That is, what new information can be gained when, rather than integrating the fluorescence signal over a photomultiplier tube, one instead retains the spatial information by using a two-dimensional video detector? In this chapter, we wish to emphasize the conceptual aspects of these experiments rather than focusing on specific issues of instrumentation and software. For a discussion of these issues the reader is referred to several comprehensive texts on this subject (Castleman, 1979; Inoue, 1986; Makovski, 1983; Taylor and Wang, 1989). In our discussion, we wish also to emphasize how certain empirical manipulations of experimental data can greatly simplify complicated analysis without sacrificing either validity or accuracy.
KeywordsFluorescence Resonance Energy Transfer Point Spread Function Frequency Space Donor Fluorescence Acceptor Fluorescence
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