Digital scanned laser light‐sheet fluorescence lifetime microscopy with wide‐field time‐gated imaging

Summary We develop a multidimensional fluorescence imaging technique by implementing a wide‐field time‐gated fluorescence lifetime imaging into digital scanned laser light‐sheet microscopy (FLIM‐DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time‐gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM‐DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.