Fluorescence lifetime imaging by time-correlated single-photon counting.
We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.
Becker
W
W
Becker & Hickl GmbH, D-12277 Berlin, Germany. becker@becker-hickl.com
Bergmann
A
A
Hink
M A
MA
König
K
K
Benndorf
K
K
Biskup
C
C
D000818:Animals; D002460:Cell Line; D004867:Equipment Design; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology; D007422:Intestines / Q000166:cytology; D007668:Kidney / Q000166:cytology; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D017785:Photons; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity