Super-multiplexing excitation spectral microscopy with multiple fluorescence bands

Fluorescence microscopy, with high molecular specificity and selectivity, is a valuable tool for studying complex biological systems and processes. However, the ability to distinguish a large number of distinct subcellular structures in a single sample is impeded by the broad spectra of molecular fluorescence. We have recently shown that excitation spectral microscopy provides a powerful means to unmix up to six fluorophores in a single fluorescence band. Here, by working with multiple fluorescence bands, we extend this approach to the simultaneous imaging of up to ten targets, with the potential for further expansions. By covering the excitation/emission bandwidth across the full visible range, an ultra-broad 24-wavelength excitation scheme is established through frame-synchronized scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF), so that full-frame excitation-spectral images are obtained every 24 camera frames, offering superior spectral information and multiplexing capability. With numerical simulations, we validate the concurrent imaging of 10 fluorophores spanning the visible range to achieve exceptionally low (∼0.5%) crosstalks. For cell imaging experiments, we demonstrate unambiguous identification of up to eight different intracellular structures labeled by common fluorophores of substantial spectral overlap with minimal color crosstalks. We thus showcase an easy-to-implement, cost-effective microscopy system for visualizing complex cellular components with more colors and lower crosstalks.