High-throughput two-photon wide-field fluorescence lifetime microscopy for dynamic membrane tension imaging

Fluorescence lifetime imaging microscopy (FLIM) offers a powerful approach to probe the physicochemical environment of biological samples with microscopic spatial resolution and independence from fluorescence intensity. In membrane biophysics, by coupling the fluorescence decay of environment-sensitive probes to local lipid packing, FLIM enables quantitative imaging of membrane mechanics in living systems. This property is exploited by the mechanosensitive probe Flipper-TR, whose fluorescence lifetime elongates as lipid packing increases upon changes in membrane tension, circumventing the limitations of conventional approaches such as tether-pulling with optical tweezers. In this thesis, we developed a two-photon excitation, wide-field, time-gated fluorescence lifetime microscope. The implementation of the rapid lifetime determination method for the single and double-exponential fluorescence decay condition, facilitated the image processing and enabled fast acquisition. A theoretical model for the fluorescence dynamics of Flipper-TR is proposed, which describes the probe population distribution across its different twisted molecular configurations and links the measured lifetime components to the underlying photophysical states. The system performance is validated on standard samples, and light-induced effects on both the biological sample and the probe are systematically characterized to define safe experimental regimes that preserve sample integrity and measurement reliability. The microscope was applied to measure membrane tension in giant unilamellar vesicles of varying lipid composition and under controlled mechanical perturbations, demonstrating sensitivity to lipid packing across distinct membrane phases. Furthermore, the system resolved the dynamic evolution of membrane tension in HeLa cells subjected to repetitive osmotic shocks, achieving a temporal resolution that surpasses that of previously reported approaches for such experiments. Together, these results establish the system as a robust platform for quantitative membrane tension imaging, and lay the groundwork for broader applications of Flipper-TR FLIM in cell biology and membrane biophysics.