A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color in vivo imaging. Its simple design, using readily available optical components, and its easy integration provide a low-cost solution for multi-color fluorescence imaging with increased efficiency and speed.