W. Becker, A. Bergmann, Lifetime-Resolved Imaging in Nonlinear Microscopy

Book Chapter, in B.R. Masters, P.T.C. So, Eds., Handbook of biomedical nonlinear optical Microscopy

The fluorescence of organic molecules is not only characterized by the emission spectrum, it has also a characteristic fluorescence lifetime. Including the fluorescence lifetime, or, more precisely, the fluorescence decay function, in the imaging process provides a direct approach to all effects involving energy transfer between different fluorophores and between fluorophores and their local environment. Typical examples are the probing of the local environment parameters of a fluorophore via lifetime changes, probing distances on the nanometer scale by FRET, and separation of fractions of the same fluorophore in different binding states to proteins, lipids, or DNA. Fluorescence lifetime imaging becomes particularly attractive in combination with multi-photon excitation. Multi-photon microscopes do not only provide the required pulsed excitation source, they also avoid crosstalk of the lifetimes in different depth of thick tissue. The chapter gives an overview on the commonly used lifetime imaging (FLIM) techniques, discusses theire pros and cons, and shows typical applications in molecular biology. The chapter ends with 209 references related to FLIM and FLIM applications.

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