Fluorescence lifetime imaging microscopy (FLIM) represents a powerful tool for biological studies. Endoscopic FLIM applied to the intracellular native biomarker NADH and FAD represents a promising mean for in vivo in situ malignant tissue diagnosis in the medical field. Else, 2-photon-excited fluorescence (2PEF) provides increased 3D resolution and imaging depth. But very few demonstrations about 2PEF lifetime measurement through a fiber have been reported and none about endoscopic 2P-FLIM through a practical fiber length (< 3m).
Our group has recently demonstrated the possibility to efficiently deliver through a very long optical fiber the short and intense excitation pulses required for 2P-FLIM. Our goal is now to check that collecting fluorescence through the same endoscopic fiber does not deteriorate the lifetime measurement. Relying on the basis previously published in case of 1PEF by P. French and co-workers (J. Biophotonics, 2015), we have experimentally quantitatively evaluated the influence on the lifetime measurement of the fiber chromatic and intermodal dispersions. The main result is that the fiber contribution to the system impulse response function, even in the case of a 3-meter long double-clad optical fiber, does not hinder the separation between free and bound NADH states using FLIM. Related calibrations and measurements will be detailed. Ongoing experiments about the development of a 2P-FLIM endomicroscope on the basis of an previously reported 2P-endomicroscope (Ducourthial et al., Sc. Reports, 2015), used under various configurations (i.e. point measurement in the center of the 2P-endomicroscope image, averaged lifetime, binned endoscopic 2P-FLIM image), will be also presented.