Multiphoton tomographic fluorescence lifetime imaging microscopy -TomoFLIM

Fluorescence lifetime imaging microscopy (FLIM) offers quantitative contrast for studying molecular interactions and cellular processes. However, its slow acquisition speeds hinder its use in dynamic biological contexts. Researchers have developed a new technique called TomoFLIM, which combines multiphoton excitation with time-correlated single-photon counting to accelerate FLIM imaging.

By using a line focus projected tomographically across the sample and time-tagged fluorescence acquisition, TomoFLIM reconstructs time-resolved data using Lucy-Richardson deconvolution and a center-of-mass method estimator. Furthermore, a physics-informed neural network called TomoFLIM Net can directly reconstruct fluorescence intensity and lifetime from compressed tomographic data.

TomoFLIM was tested against traditional raster-scanned FLIM measurements using calibrated beads and biological samples. The results showed that TomoFLIM achieved compression ratios over 90% and a Pearson correlation coefficient above 80% compared to reference images. Importantly, the technique was 16 times faster than raster-scanned FLIM, with a 3.75-second runtime for a dataset previously acquired in 60 seconds.

TomoFLIM Net successfully recovered distinct lifetime populations from experimental beads, demonstrating the potential for rapid live-cell imaging of dynamic biological processes, even within turbid specimens.