Journal
MICROSCOPY RESEARCH AND TECHNIQUE
Volume 69, Issue 3, Pages 186-195Publisher
WILEY
DOI: 10.1002/jemt.20251
Keywords
time-correlated single photon counting (TCSPC); fluorescence lifetime imaging (FLIM); Forster resonance energy transfer (FRET); autofluorescence; fluorescence correlation spectroscopy (FCS)
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Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.
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