Two-photon lifetime imaging of fluorescent probes in intact blood vessels: A window to sub-cellular structural information and binding status

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by similar to 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.