Image analysis for denoising full‐field frequency‐domain fluorescence lifetime images

Video‐rate fluorescence lifetime‐resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full‐field frequency‐domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video‐rate image acquisition is possible. Homodyne FLIM uses a gain‐modulated image intensified charge‐coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super‐resolution imaging).