Rapid fluorescence lifetime imaging microscopy via few-photon imaging

Conventional fluorescence lifetime imaging microscopy (FLIM) based on time-correlated single photon counting has great potential in various domains, notably in cellular biology, enabling comprehensive studies encompassing spatiotemporal dynamics and quantitative analysis of fluorescence lifetimes. However, it usually requires a long acquisition time, which limits its application in rapid imaging scenarios, such as investigation of rapidly evolving biological events and observation of living organisms. This work proposes a rapid fluorescence lifetime imaging scheme, which reduces the requirement of photon accumulating number and enables rapid fluorescence lifetime estimation under photon-limited conditions. Instead of relying on accumulated photons, the proposed scheme records the counts of emitted laser pulses upon photon counting events within different time gates to estimate the fluorescence intensity, and the fluorescence lifetime is then calculated using the rapid lifetime determination algorithm. Experimental results demonstrate that in order to reconstruct fluorescence lifetime images with similar quality, the proposed scheme requires only a fifth acquisition time that of the conventional time-correlated single photon counting FLIM. The proposed method offers a potential possible approach for rapid fluorescence lifetime determination in dynamic applications.