High-throughput hyperspectral fluorescence imaging using a high-speed silicon photomultiplier array

High-speed multiplex imaging of fluorescent probes is limited by a combination of spectral resolution, sensitivity, high cost, and low light throughput of detectors and filters. In this work, we present a hyperspectral detection system based on a silicon photomultiplier array that enables high-speed, high-light-throughput hyperspectral imaging at low cost. We demonstrate 16 spectral channel imaging at 50 MP/s (800M spectra per second) with a conventional two-photon microscope combined with a generalized spectral unmixing model that enables the extraction of spectrally overlapping fluorophores. We show that the high spectral resolution combined with high detection throughput enables the multiplexing of multiple contrast agents as well as the detection of subtle spectral shifts associated with molecular binding. Silicon photomultiplier arrays may be a promising method to extend multiplex fluorescence imaging in a variety of scenarios.