We discuss our recent efforts to achieve highly multiplexed fast fluorescence imaging and quantitative biosensing through camera frame-synchronized scanning of the excitation wavelength in the wide field. For live-cell microscopy, we thus attain low (~1%) crosstalks and ~10 ms temporal resolutions for up to six fluorophores via linear unmixing, and further develop novel, quantitative imaging schemes for both bi-state and FRET fluorescent biosensors. These capabilities are further integrated to multiplex absolute pH imaging with three additional target proteins in the mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging without fluorescence dispersion.
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