Complex wavelet filter improves FLIM phasors for photon starved imaging experiments

Abstract

Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores’ lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM phasor analysis. This filtering strategy preserves fine structures and reports accurate lifetimes in photon starved FLIM imaging. Moreover, this filter outperforms median filters and makes FLIM imaging with lower laser power and faster imaging possible.