A readily usable two-photon FLIM microendoscope.

A two-photon fluorescence lifetime (2P-FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular 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 is 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 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. This article is protected by copyright. All rights reserved.