Abstract
In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.
Highlights
IntroductionNADH have a free lifetime component of ≈0.4 ns and an enzyme-sensitive bound one of 2.8 to 3.4 ns [4,5,6]
Malignant tissue diagnosis would greatly benefit from a flexible in vivo in situ label-free and minimally invasive high resolution imaging tool, capable of several hundreds of microns imaging depth to address a typical epithelium [1]
For the first time to our knowledge, nonlinear endogenous fluorescence lifetime imaging microscopy (FLIM) in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs)
Summary
NADH have a free lifetime component of ≈0.4 ns and an enzyme-sensitive bound one of 2.8 to 3.4 ns [4,5,6]. The higher rate of glycolysis induces a higher free/enzyme-bound ratio and a variation of key-enzyme activity typically associated with a decrease of the longlifetime component, leading to a faster fluorescence decay. Assessing this faster decay has enabled to discriminate between normal and tumor tissues [7,8,9] and cells [6]. Fluorescence lifetime has the advantage, over intensity, of its immunity to concentration and quantum yield variations which makes lifetime imaging adapted to in vivo in situ imaging
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