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Methods Enzymol. 2003;360:509-42.

Fluorescence lifetime-resolved imaging: measuring lifetimes in an image.

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  • 1Department of Physics, University of Illinois Urbana-Champaign, Urbana 61801, USA.


We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation. Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation--and therefore affect the measured fluorescence lifetime--that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging. FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging extends considerably the potential information that can be derived from a fluorescence experiment. Our purpose has been to arouse an appreciation for the broad application of fluorescence lifetime-resolved measurements in imaging. We have given only general design characteristics of the instrumentation and discussed the characteristics that distinguish imaging from the single channel lifetime-resolved measurements. We have not provided details of the instrumentation or the presented many examples. These are available in the literature, and given in the references, and they are continually and rapidly growing.

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