title: FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy.
abstract: The combination of fluorescent-probe technology plus modern optical microscopes allows investigators to monitor dynamic events in living cells with exquisite temporal and spatial resolution. Fluorescence recovery after photobleaching (FRAP), for example, has long been used to monitor molecular dynamics both within cells and on cellular surfaces. Although bound by the diffraction limit imposed on all optical microscopes, the combination of digital cameras and the application of fluorescence intensity information on large-pixel arrays have allowed such dynamic information to be monitored and quantified. Fluorescence lifetime imaging microscopy (FLIM), on the other hand, utilizes the information from an ensemble of fluorophores to probe changes in the local environment. Using either fluorescence-intensity or lifetime approaches, fluorescence resonance energy transfer (FRET) microscopy provides information about molecular interactions, with Ångstrom resolution. In this review, we summarize the theoretical framework underlying these methods and illustrate their utility in addressing important problems in reproductive and developmental systems.
authors.affiliation: Department of Bioengineering, University of California, Riverside, California.
authors.forename: Carla
authors.initials: C
authors.lastname: De Los Santos
authors.affiliation: Department of Electrical Engineering, Stanford University, Stanford, California.
authors.forename: Ching-Wei
authors.initials: CW
authors.lastname: Chang
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan.
authors.forename: Mary-Ann
authors.initials: MA
authors.lastname: Mycek
authors.affiliation: Department of Bioengineering, University of California, Riverside, California.|Department of Biology, University of California, Riverside, California.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cardullo
mesh_terms: D000818:Animals; D036681:Fluorescence Recovery After Photobleaching / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods