Multispectral multiplexed confocal FLIM for live cell imaging

Spectrally resolved fluorescence lifetime imaging (FLIM) with high spatial precision offers comprehensive information on species localization and behavior. It is challenging to resolve weak fluorescence signals in multiple dimensions (spatial, spectral, and temporal) at high frame rates, especially in dynamic live cell processes, as photobleaching and phototoxicity limit acceptable photon count rates. We developed a multiplexed confocal FLIM technique, which uses a prism-based imaging spectrometer to separate a 10 × 10 array of confocal foci into their spectral components. This allows the sampling of the spectra by a time-resolved image sensor to produce a multispectral time-resolved data set used for generating multispectral lifetime images. This system captures 300 × 300 pixel FLIM containing 12 unique spectral bands covering a 450–700 nm spectral range in 1.8 s of exposure. Its performance was demonstrated in fixed stained samples and in multispectral imaging of FLIM-FRET in live cells.