Fluorescence Lifetime Imaging Microscopy

Fluorescence lifetime imaging microscopy (FLIM) produces spatially resolved images of fluorophore lifetime (the property describing how rapidly fluorescence decays), providing another dimension of information for visualizing fluorophores and an additional source of contrast. The basics of fluorophore lifetime, including the characteristics of fluorophore decay, a discussion of factors affecting fluorescence lifetimes, and the usefulness of lifetimes as a source for contrast. Both time domains (TD) and frequency-domain (FD) approaches (including data analysis) are described along with an introduction to different microscopy methods for lifetime imaging. Various applications of FLIM, ranging from commonly used fluorescence resonance energy transfer (FRET)-FLIM to fluorescence correlation spectroscopy (FCS)-, MS-MP-, and video-rate FLIM are also outlined in this chapter. Continuing advances in technology for microscopy and the developing appreciation that fluorescence lifetime is a sensitive means for evaluating microenvironment will likely help make FLIM a critical research tool for cell biology by providing a new way for cell biologists to detect, visualize, and investigate structure and function in biological systems.