Modulated Excitation Spectral Microscopy: Superior Unmixing and Multiplexed Imaging with Minimal Excitation Wavelengths

Excitation spectral microscopy enables multitarget imaging with high temporal resolution but is fundamentally constrained by a long-standing physical limitation: the number of imaging targets cannot exceed the number of excitation wavelengths due to the requirement for spectral unmixing. Here, we introduce modulated excitation spectral microscopy (MoExSM), which overcomes this constraint by modulating the excitation intensity of each wavelength in a specific ratio for each frame and decoupling the excitation and acquisition channels. MoExSM enables multiplexed imaging of up to five subcellular targets in fixed cells by using only three excitation wavelengths and a single, fixed fluorescence emission detection band, achieving minimal crosstalk. In live-cell imaging, it allows visualization of complex and dynamic interactions among four organelles, excited by three wavelengths. By breaking the physical limitation imposed by excitation wavelengths and simplifying the system design, MoExSM offers tremendous potential for visualizing complex cellular components with more colors.