Three-dimensional live multi-label light-sheet imaging with synchronous excitation-multiplexed structured...

Area of Science:

• Biophysics
• Microscopy
• Optical Imaging

Background:

• Multiplexed imaging is crucial for understanding complex biological interactions.
• Live imaging requires high speed and resolution, posing challenges for spectral imaging.
• Existing methods may compromise speed or resolution when capturing multiple fluorescent markers.

Purpose of the Study:

• To introduce spectral-encoded structured illumination (spectral-SIM) light-sheet microscopy.
• To enable parallel, multi-excitation-channel 3D imaging.
• To achieve synchronous imaging without sacrificing speed or resolution.

Main Methods:

• Spectral-SIM encodes excitation wavelength into the illumination pattern's phase.
• Synchronous image capture across multiple excitation channels is performed.
• Structured light-sheet microscopy principles are retained for background removal.

Main Results:

• Achieved parallel multi-excitation-channel imaging in 3D.
• Maintained imaging speed and spatial resolution comparable to mono-channel imaging.
• Generated clear 3D multiplexed images in thick tissues, demonstrating deep imaging capabilities.

Conclusions:

• Spectral-SIM light-sheet microscopy is a powerful tool for live, multiplexed 3D imaging.
• The technique overcomes limitations of previous spectral imaging methods.
• Demonstrated successful deep tissue imaging in live zebrafish with high resolution.