FLIM and FCS detection in laser-scanning microscopes: increased efficiency by GaAsP hybrid detectors.
Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less-than-ideal signal-to-noise ratio of the lifetimes obtained: either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10⁻³ mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal-dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS.
Becker & Hickl GmbH, Nahmitzer Damm 30, Berlin, Germany. becker@becker-hickl.de
W
W
Becker
B
B
Su
O
O
Holub
K
K
Weisshart
D000818:Animals; D018613:Microscopy, Confocal / Q000295:instrumentation*; D012867:Skin / Q000737:chemistry / Q000166:cytology*; D013552:Swine
FCS; FLIM; confocal microscopy; hybrid detector; multiphoton microscopy