Two-Photon Holographic Microscopy to Study Neural Activity and Connectivity in a Mouse Model

Begin with an awake mouse positioned under a two-photon holographic microscope integrated with spatial light modulators or SLMs.

The mouse holds a pre-implanted head plate.

In the mouse brain, neurons express red-light-sensitive channels and green-fluorescent calcium indicators that bind to calcium ions.

To study neural activity and connectivity, set the imaging parameters.

Use a 920 nanometers laser to excite calcium-bound indicators within the neurons. Capture the neuron's fluorescence image with minimal photodamage.

Reset the imaging parameters and focus a near-infrared laser beam.

SLMs shape the laser beam into precise holographic patterns that allow selective focusing on target neurons at multiple points.

This stimulates light-sensitive channels, causes calcium ion influx, and enhances the target neurons' fluorescence.

The activated target neurons transmit signals to the connected neurons and increase the connected neuron's fluorescence.

Again, capture a two-photon image. An increased fluorescence in target and connected neurons confirms neuronal activity and connectivity.

After placing the mouse under the microscope and performing two-photon imaging, open the commercial imaging software. In the live imaging mode, adjust the voltage of the image detector and the power of the imaging laser, to optimize the brightness of the neurons expressing GCaMP6m-P2A-ChRmine. Capture images of the neurons expressing these proteins, and then illuminate the specific neurons following the procedure shown earlier.

To investigate the functional connectivity within layer 2 and 3 neurons, use a spatial light modulator to generate holographic patterns of optogenetic stimulation. And combine it with two-photon calcium imaging. Set the intensity of the imaging laser to 920 nanometers at 10 to 20 milliwatts, and the field of view to 256 micrometers by 256 micrometers. Measured at a depth of 100 to 150 micrometers from the cortical surface.

Set the pixel dwell time at 1.5 microseconds for 2 Hertz, or 100 nanoseconds for 30 Hertz. Use both 2 Hertz and 30 Hertz as the imaging frame rate to see if a single holographic stimulus caused a calcium response in the neurons. Set the intensity of the holographic stimulation laser that stimulates a single neuron at 10 milliwatts, which is sufficient to induce neural activity.

Simultaneously, image the calcium ion response at 920 nanometers, with 10 holographic stimuli at 1,040 nanometers and eight seconds intervals for a duration of 50 milliseconds after a baseline period of 10 seconds.