Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.
We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.
Kumar
S
S
Dunsby
C
C
De Beule
P A A
PA
Owen
D M
DM
Anand
U
U
Lanigan
P M P
PM
Benninger
R K P
RK
Davis
D M
DM
Neil
M A A
MA
Anand
P
P
Benham
C
C
Naylor
A
A
French
P M W
PM