Less Can Be More: Optimization of the Acquisition Range to Enhance Phasor Analysis in Fluorescence Lifetime Imaging Microscopy

The phasor approach is widely recognized as a powerful method for analyzing multidimensional images, and is currently one of the most utilized algorithms for spectral unmixing in fluorescence microscopy. Notably, it is exstensively employed in fluorescence lifetime imaging microscopy, but limitations in the acquisition window can affect the accuracy of lifetime and fraction estimates if not properly considered. In this article, we introduce a novel analytical framework to predict how photophysical and acquisition parameters influence the values of lifetimes and fractions estimated via phasor analysis. This approach also accounts for photon loss during detection, allowing researchers to mitigate errors by optimizing integration bounds. By refining these parameters, we enhance the precision of lifetime estimation and improve fluorescence signal clarity, particularly for low-signal contributions in autofluorescence imaging. Our findings expand the utility of phasor analysis across fluorescence microscopy techniques, even in challenging conditions where capturing the full emission spectrum is difficult. This advancement improves the specificity and sensitivity of fluorescence microscopy, providing more accurate quantification and separation of fluorophore contributions in complex biological samples.