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.