Fig. 4: Fluorescence lifetime image-scanning microscopy in live cells.
From: The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
a Intensity-based ISM image and b lifetime-based FLISM image of live HeLa cells stained with the polarity-sensitive fluorescent probe di-4-ANEPPDHQ. Side images depict the areas within the dashed white boxes. c Histogram distribution of the fluorescence lifetime values (top): number of pixels versus lifetime values. Pixel intensity thresholded phasor plots (bottom): number of pixels versus the polar coordinate (10% thresholds). d Phasor-based segmentation, i.e, images obtained by backprojection of points within the red (long lifetime, ordered membrane) and green (short lifetime, disordered membrane) area. Scale bars 5 μm. Pixel dwell time 150 μs. While here we show one cell, this experiment was independently performed two times on the same HeLa cell line. Setup: custom-built single-photon laser-scanning microscope equipped with a 5 × 5 SPAD array detector prototype.
