Multiphoton tomographic fluorescence lifetime imaging microscopy -TomoFLIM

Abstract

Fluorescence lifetime imaging microscopy provides quantitative, concentration-independent contrast for probing molecular interactions, biochemical environments and cellular physiology. However, the requirement to acquire sufficient time-resolved photon statistics makes FLIM inherently slow, limiting its application to dynamic biological processes. In live-cell applications, including calcium signalling and vesicular trafficking, acquisition times can exceed the timescale of the underlying biology, causing temporal averaging, motion artefacts and loss of transient information. Methods that increase FLIM acquisition speed while preserving quantitative lifetime accuracy are therefore required. We report on a high-speed, compressive, multiphoton fluorescence lifetime imaging technique (TomoFLIM). Two-photon fluorescence is excited using a line focus projected tomographically across the sample, while time-tagged fluorescence is acquired using time-correlated single-photon counting. Time-resolved fluorescence data are reconstructed using Lucy-Richardson deconvolution followed by a computationally efficient centre-of-mass method lifetime estimator. In addition, TomoFLIM Net, a physics-informed neural-network, directly reconstructs fluorescence intensity and lifetime from compressed time-resolved tomographic data. TomoFLIM was benchmarked against raster-scanned fluorescence lifetime measurements using calibrated fluorescence lifetime beads and biological specimens. We demonstrate imaging at compression ratios exceeding 90%, with Pearson correlation coefficients above 80% relative to reference images. A raster-scanned FLIM dataset acquired in 60 s was reproduced using TomoFLIM in 3.75 s, representing a 16-fold increase in frame rate and equivalent reduction in accumulated dark counts. TomoFLIM Net recovered distinct experimental bead lifetime populations, demonstrating a direct route from compressed measurements to quantitative lifetime maps. TomoFLIM therefore offers significant potential for rapid live-cell imaging of dynamic biological processes, including deep within turbid biological specimens.