ROI-summed analysis for improved fluorescence lifetime fitting accuracy and precision

Laser-scanning multiphoton microscopy with time-correlated single photon counting (TCSPC) enables fluorescence lifetime imaging microscopy (FLIM) with high spatial and temporal resolution, albeit with limited imaging speed. FLIM images are typically analyzed at the pixel level by fitting pixel decays to multiexponential decay models and assigning lifetimes. Yet, often, only cell-level lifetime parameters are used in statistical analyses. Therefore, pixel-level lifetime parameters are averaged over the cell mask to produce single-cell lifetime parameters. This analysis pipeline is inefficient and introduces variability and bias to the results. We characterize fitting the aggregate decay from all cell pixels as a more accurate and precise method of obtaining cell-level lifetime statistics with shorter integration times.