Fluorescence lifetime imaging microscopy (FLIM).
Fluorescence lifetime imaging microscopy (FLIM) is a technique to map the spatial distribution of nanosecond excited state lifetimes within microscopic images. FLIM systems have been implemented both in the frequency domain, using sinusoidally intensity-modulated excitation light and modulated detectors, and in the time domain, using pulsed excitation sources and time-correlated or time-gated detection. In this review we describe the different modes in which both frequency-domain and time-domain FLIM instruments have been constructed in wide-field and in point-scanning (confocal) microscopes. Also, novel additional strategies for constructing FLIM-instruments are discussed. In addition to technical implementation, this chapter gives an overview of the application of FLIM in cell biological en biomedical studies. Especially for in situ protein-protein interaction studies using fluorescence resonance energy transfer (FRET), FLIM has proven to be a robust and established technique in modern cell biology. Other application areas, including usage of lifetime contrast for ion-imaging, quantitative imaging, tissue characterization and medical applications, are discussed.
van Munster
Erik B
EB
Swammerdam Institute for Life Sciences & Centre for Advanced Microscopy, Section Molecular Cytology, Kruislaan 316, 1098 SM Amsterdam, The Netherlands. munster@science.uva.nl
Gadella
Theodorus W J
TW
D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*
D011506:Proteins