Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Begin with a transgenic mouse secured in a stereotactic frame, with a cranial window implanted over its visual cortex.

The mouse expresses EGFP in the microglia and R-CaMP, a red fluorescent calcium indicator, in the neurons of the visual cortex.

Position the mouse under a two-photon microscope and adjust the objective lens to focus on the brain surface.

Move the mouse with the stereotactic frame from the objective lens and attach a shading device.

Fill the shading device with water and reposition the mouse under the microscope.

Cover the lens with black foil.

The shading device and foil block external light interference, ensuring accurate imaging.

Place an LCD monitor in front of the mouse’s eye to provide visual stimulation.

Set the imaging parameters and begin imaging.

Visual stimulation triggers neuronal excitation, leading to calcium influx and an increase in R-CaMP fluorescence.

Meanwhile, microglia retract their processes while interacting with activated neurons, as observed through live imaging.

To install the custom-made shading device, and an LCD monitor for visual stimulation, first, position the lens to focus on the brain surface. And then set this lens position as the original z position. Keep the x, y-coordinates constant and elevate the objective lens. Remove the mouse and stereotaxic instrument from the objective lens. Attach a shading device on the top of the head plate using silicone, ensuring that the space between the head plate and the shading device is well sealed.

Fill the shading device with distilled water. Then, fix the mouse with the stereotaxic frame under the objective lens. Carefully reset the focal plane at the brain surface, checking the depth of the objective lens. Cover the objective lens with black aluminum foil to avoid light contamination from the LCD monitor. Set a 10-inch LCD monitor at 12.5 centimeters in front of the eyes of the mouse to present visual stimuli.

Configure the fluorescence emission collection filters for EGFP and RCaMP, and the excitation wavelength to 1,000 nanometers. Acquire images with a spatial resolution of 0.25 microns per pixel. Find the imaging region where RCaMP-positive neurons and EGFP-positive microglia can be simultaneously imaged in Layer 2/3.

Acquire images at the frame rate of 30 Hertz. Simultaneously, with the image acquisition, present drifting grating visual stimuli to the mouse in 12 directions at 6 orientations from 0 to 150 degrees in 30-degree steps.