A readily usable two-photon fluorescence lifetime microendoscope.

A two-photon fluorescence lifetime (2P-FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub-cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table-top microscope performances are achieved through a comprehensive system including a home-designed spectro-temporal pulse shaper and a custom air-silica double-clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.