Abstract
A spatial-spectral multiplexing two-photon excited fluorescence (TPEF) hyperspectral imaging system was demonstrated at 17 frames per second (fps) with over 2,000 effective spectral channels. The system exploited the hardware used in beam-scanning microscopy to perform multiplexing of spatial and spectral information, leading to two orders of magnitude reduction in data throughput. An iterative demultiplexing algorithm enabled robust classification and full spectral recovery without requiring prior knowledge of the emission spectra of the fluorophores. Proof-of-concept demonstrations were performed using spatially heterogeneous multicomponent laser dyes and live genetically encoded fluorescent C. elegans. This instrument requires minimal hardware modifications for most existing multiphoton microscope and provides the ability to acquire hyperspectral TPEF images at video rate. It has the potential for dynamic studies in deep tissue with multiple spectrally overlapping fluorescence tags.
Published Version
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