Sub-100 nm 3-D fluorescence lifetime imaging using time correlated single photon counting detection and multifocal multiphoton excitation

The interaction of matter and light is one of the fundamental processes occurring in nature, and its most elementary form is realized when a single atom interacts with a single photon. Reaching this regime has been a major focus of research in atomic physics and quantum optics for several decades and enables fascinating applications such as 3-D fluorescence imaging. Here 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.