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

摘要

The interaction of matter and light is one of the fundamental processesoccurring in nature, and its most elementary form is realized when a singleatom interacts with a single photon. Reaching this regime has been a majorfocus of research in atomic physics and quantum optics for several decades andenables fascinating applications such as 3-D fluorescence imaging. Here wereport a multifocal multiphoton time-correlated single photon counting (TCSPC)fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channelmulti-anode PMT detector. Multiphoton excitation minimizes out-of-focusphotobleaching, multifocal excitation reduces non-linear in-planephotobleaching effects and TCSPC electronics provide photon-efficient detectionof the fluorescence decay profile. TCSPC detection is less prone to bleaching-and movement-induced artefacts compared to wide-field time-gated orfrequency-domain FLIM. This microscope is therefore capable of acquiring 3-DFLIM images at significantly increased speeds compared to single beammultiphoton microscopy and we demonstrate this with live cells expressing a GFPtagged protein. We also apply this system to time-lapse FLIM of NAD(P)Hautofluorescence in single live cells and report measurements on the change inthe fluorescence decay profile following the application of a known metabolicinhibitor.