Multispectral live-cell imaging with uncompromised spatiotemporal resolution

Abstract Multispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four, but standard approaches are poorly suited to live-cell imaging. We introduce an approach for multispectral imaging in live cells, comprising an iterative spectral unmixing algorithm and eight channel camera-based image acquisition hardware. This enables the accurate unmixing of low signal-to-noise ratio datasets, typical of live-cell imaging, captured at video rates. We use this approach on a commercial spinning disk confocal microscope and a home-built oblique plane light sheet microscope to image 1–7 spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions and small-molecule dyes. We further use de novo designed protein-binding proteins (minibinders), labelled with organic fluorophores, and use these in combination with our multispectral imaging approach to study the endosomal trafficking of cell-surface receptors at endogenous levels.