A compact holographic projector module for high-resolution 3D multi-site two-photon photostimulation

Area of Science:

• Neuroscience
• Biophysics
• Optical Engineering

Background:

• Two-photon (2P) photolysis with holographic illumination allows simultaneous 3D stimulation of neuronal networks.
• Limited field-of-view (FOV) and resolution of holographic projectors restrict detailed analysis of dendritic integration.

Purpose of the Study:

• To overcome the FOV limitations of holographic 2P photolysis.
• To enable precise, simultaneous stimulation of multiple dendritic spines within a larger imaging area.

Main Methods:

• Integrated a holographic projector with a commercial 2P galvanometer scanning microscope.
• Utilized galvanometer mirrors to reposition the holographic FOV across the imaging FOV.
• Developed custom software for 3D positioning of uncaging sites within acquired image stacks.

Main Results:

• Extended the accessible holographic FOV within a commercial 2P microscope.
• Demonstrated simultaneous glutamate uncaging at multiple dendritic spines.
• Observed summation of postsynaptic potentials leading to action potentials.
• Enabled simultaneous 2P Ca2+ imaging to monitor synaptic Ca2+ entry and dendritic events.

Conclusions:

• The developed system overcomes FOV limitations for high-resolution patterned 2P photolysis.
• This technique facilitates detailed investigation of synaptic integration and dendritic computation.
• The system allows simultaneous stimulation and imaging, providing comprehensive insights into neuronal function.