Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.

Time-correlated single-photon counting (TCSPC) is the gold standard for performing lifetime spectroscopy in biological assays. Traditional fluorescence lifetime imaging (FLIM) using laser scanning microscopes are inherently slow due to point scanning all pixels in the field-of-view. Wide-field implementations of TCSPC spectroscopy using microchannel plates benefit from particularly fast acquisition times at the expense of temporal resolution, and are fundamentally limited by photon counting rates.

Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors.