Multiplexed imaging is a powerful tool for studying complex interactions inside biological systems. Spectral imaging methods that capture multiple fluorescent markers synchronously without sacrificing the imaging speed or resolution are most suitable for live imaging. We describe spectral-encoded structured illumination (spectral-SIM) light-sheet microscopy, which enables parallel multi-excitation-channel imaging in 3D. Spectral-SIM encodes the excitation wavelength as the phase of the illumination pattern, and allows synchronous image capture over multiple excitation channels at the same speed and spatial resolution as mono-channel structured light-sheet imaging. The technique retains structured light-sheet microscopy’s ability in removing out-of-focus and scattered emission background, and generates clear 3D multiplexed images in thick tissue. The capability of this technique was demonstrated by the imaging of live triple-labeled transgenic zebrafish to over 300 μm deep with 0.5μm-by-2μm (lateral-by-axial) resolution.
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