Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications

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Abstract
The new and novel techniques of fluorescence lifetime imaging (FLI)∗∗ and fluorescence lifetime imaging microscopy (FLIM) provide the investigator with the capacity to quantitate two-dimensional fluorescence intensity distributions and lifetimes. The concept, theory, and instrumentation of FLI and FLIM are reviewed in this paper. The implementation of FLIM instrumentation with conventional and confocal microscopic systems is discussed. These instruments permit the quantitative measurement of molecular interactions and chemical environment from samples in biological, physical, and environmental sciences. Numerous applications in the biomedical sciences for FLIM instrumentation are also discussed. ∗∗ We refer to the measurement of fluorescence lifetime images for macrosamples (e.g., cuvette) without use of a microscope as fluorescence lifetime imaging (FLI). whereas measurements obtained with a microscope are termed fluorescence lifetime imaging microscopy (FLIM).