#32 · Single-cell

Computational readouts of calcium transients and FLIM lifetime artifacts in microscopy

The dominant thread is quantitative extraction of subcellular measurements from fluorescence images, where background, point-spread overlap, or event detection must be handled explicitly. Astro-BEATS transfers astronomical methods, Rolling Hough Transform background estimation and DBSCAN source finding, to segment miniature synaptic calcium transients. A three-channel FLIM-FRET approach with a hierarchical EM estimator quantifies per-cell, membrane-proximal alpha-synuclein aggregation in live neurons, with validation by Monte Carlo simulation. A separate study shows that MSSR-based intensity masks applied before phasor FLIM analysis suppress PSF-overlap lifetime blending in U2OS cells and in simulations. What is new is the application of each method to a specific readout: event-level calcium transients, per-cell aggregation in live neurons, and lifetime estimates corrected for PSF overlap.