Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Area of Science:

• Neuroscience
• Biophotonics
• Microscopy

Background:

• Multiphoton microscopy, including two-photon microscopy (2PM), is crucial for subcellular resolution in vivo imaging.
• Three-photon microscopy (3PM) offers advantages for deep-tissue imaging over 2PM, utilizing longer wavelength lasers and higher-order nonlinear excitation for reduced scattering and background.

Purpose of the Study:

• To optimize a three-photon microscopy (3PM) setup for deep-tissue in vivo imaging.
• To provide a protocol for biology laboratories, especially those with existing two-photon microscopy (2PM) systems, to implement 3PM.
• To demonstrate the capabilities of 3PM for high-contrast structural and functional imaging in intact mouse and zebrafish brains.

Main Methods:

• Optimization of a typical three-photon microscopy (3PM) setup.
• Prechirping of ~1,300 nm and ~1,700 nm laser pulses.
• Animal preparation and intravital 3D imaging protocols for mice and zebrafish.

Main Results:

• Three-photon microscopy (3PM) achieves high-contrast imaging deep within scattering tissues like the mouse brain (cortex to hippocampus, entire forebrain) and adult zebrafish.
• Commercial laser sources and microscopes make 3PM readily available for biological research.
• Successful demonstration of intravital 3D imaging in intact mouse and adult zebrafish brains using the optimized 3PM protocol.

Conclusions:

• Three-photon microscopy (3PM) significantly enhances deep-tissue in vivo imaging capabilities compared to 2PM.
• The availability of commercial components and optimized protocols facilitates the adoption of 3PM in biological research laboratories.
• 3PM provides a powerful tool for high-resolution, deep-brain imaging in preclinical models.