Figure 4.

Preparing the micropipette before injection

(A) The presence of beads is verified by making a puff above the tissue (green arrowhead).

(B) The dextran cloud is visualized to determine flow rate, also above the tissue (yellow arrowhead marking the end of the micropipette).

(C) Micropipette is lowered until it touches the tissue surface (yellow arrowhead marking end of tip).

(D) Image of tissue at a depth of 50 μm below the surface, where only one section of the pipette is visible (yellow arrowhead).

(E) Example time course from Zeiss software over the Region of Interest (ROI) placed onto the image. The red circle (right) displays where the ROI is located.

The time graph on the left shows the changes in all three channel fluorescence intensities within that ROI over the 2 h.

The yellow arrowhead shows the peaking of dextran intensity during the initial injection(s).

The blue arrowhead shows that the microglial process present right around the end of the micropipette was briefly blown away at the time of injection (seen through the drop in green intensity).

The pink arrowhead demonstrates when the red beads were deposited into the tissue. The green arrowhead shows injection times.

In this case, there were 3 injections needed to deposit the correct number of beads into the tissue. The orange arrowhead is looking at the endpoint fluorescence intensity of the three channels.

The blue line for the dextran has returned to its baseline value before injection.

The red line has stayed the same since injection, pointing towards the beads staying in focus and not drifting.

The green line has steadily increased until reaching this timepoint, alluding to process interaction with beads and increased microglial process presence in the ROI.