Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

Place a mouse brain slice in a perfusion chamber containing aCSF under a two-photon microscope.

The mouse is genetically engineered to express GFP in microglia, enabling microglial movement tracking.

Secure the slice with a holder.

Using bright-field illumination, locate the region of interest.

Switch to fluorescence illumination to visualize the microglia. 

Fill a micropipette with the test compound, mount it on a syringe holder, and lower it to touch the slice.

Activate two-photon imaging mode, focusing low-energy near-infrared light into the tissue.

At the laser’s focal point, two photons combine their energy to excite GFP, emitting fluorescence.

Capture images at multiple Z-planes to focus on the microglial processes.

Record baseline activity, then inject the compound and continue recording.

The compound binds to microglial receptors, triggering signaling pathways that drive microglial process extension toward the compound source.

Monitor the fluorescence increase near the injection site over time to track the movement.