Holographic two-photon activation for synthetic optogenetics.

Area of Science:

• Neuroscience
• Optogenetics
• Molecular Biology

Background:

• Optogenetics enables photocontrol of cell activity using microbial opsins.
• Synthetic optogenetics photocontrols mammalian proteins using photoswitches and genetic modification.
• Visible light's poor tissue penetration limits synthetic optogenetics in vivo, often requiring two-photon excitation (2PE).

Purpose of the Study:

• To develop an alternative synthetic optogenetics technique using 2PE-compatible photoswitches.
• To enable photoactivation of genetically modified glutamate receptors.
• To facilitate fast, multi-region neural photoactivation using 2P-digital holography (2P-DH).

Main Methods:

• Utilized 2PE-compatible photoswitches for targeted photoactivation.
• Engineered glutamate receptors for photocontrol.
• Implemented 2P-digital holography (2P-DH) for spatiotemporal control.
• Integrated 2P-DH with synthetic optogenetics and electrophysiology or Ca2+ imaging for all-optical interrogation.

Main Results:

• Demonstrated successful photoactivation of genetically modified glutamate receptors using 2PE-compatible photoswitches.
• Achieved fast, multi-region neural photoactivation via 2P-DH.
• Successfully combined 2P-DH and synthetic optogenetics with downstream recording techniques.

Conclusions:

• Developed a novel synthetic optogenetics approach compatible with 2PE.
• Enabled precise, deep-tissue photocontrol of neuronal activity.
• Provided a robust platform for all-optical neural circuit interrogation.