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.

30 minutes before starting the recording, connect the peristaltic pump to a customized recording chamber with top and bottom perfusion for optimizing the oxygenation and viability of the tissue slices and clean the entire perfusion system with 50 milliliters of ultrapure water. At the end of the cleaning cycle, start the perfusion of the recording chamber with 50 milliliters of aCSF in a glass beaker under constant carbogenation and use a disposable wide-mouth transfer pipette to transfer the first slice to be imaged to the beaker to remove the lens paper.

When the section has sunk to the bottom of the beaker, transfer the section to the recording chamber and position the slice holder onto the slice to minimize movement induced by the perfusion flow. Use brightfield illumination to target the brain region of interest under a 5 to 10 times magnification. Then use a 25 times objective with a 0.35 times water immersion lens to adjust the position of the viewing field.

Use fluorescence illumination to locate the fluorescent microglial cells and backfill the pipette with 10 microliters of aCSF containing the compound of interest at its final concentration. Point the tip downward with gentle shaking to remove any air bubbles trapped in the tip and mount the filled pipette into a pipette holder connected to a 5-milliliter syringe, with a plunger positioned at the 5-milliliter position and mounted onto a three axis micromanipulator.

Lower the pipette gently toward the slice, controlling and adjusting the objective at the same time, until the pipette tip lightly touches the surface of the slice. Now tune the laser and switch the microscope to the multiphoton mode. Make sure that the chamber is screened from any light source and switch on the non-descanned detectors. Set the gain and use a lookup table with a color-coded upper limit to avoid saturating the pixels in the image.

Then start recording for a total duration of at least 30 minutes, slowly depressing the syringe plunger from the 5 to 1 milliliter position after five minutes over a period of 5 seconds to apply the compound to the section. For image analysis, first perform Z-projection and drift correction with ImageJ on the file of interest. Then open the modified file in Icy and draw a circular, 35-micrometer diameter region of interest centered over the injection site.

Run the movie again with the ROI to ensure that it is well-positioned. Then, use the region of interest Intensity Evolution plugin to measure the mean intensity over time in the region of interest and save the results as a .xls file.