(A–E) XY-precision of photostimulation. (A) Image of surface vessels labeled with Texas Red-dextran in an opsin mouse that were photostimulated with spots in different positions relative to the vessel wall. Dashed rectangle indicates region in inset on the difference image panels (B–D). Three spot positions were used: (B) spots centered on vessel walls, (C) spot edges on vessel walls, and (D) spot centers one diameter away from vessel walls. Difference images as in Figure 2. Red circles indicate the location and size of the spots, white asterisks indicate spot centers. (E) Time courses of diameter changes based on spot position. Average of three vessels, five repetitions each. Error bands are standard deviation (SD). Total laser power was ~130 mW divided between the six spots. Mean constriction for the three conditions was 7.6%, 4.8%, and 0.0% for B, C, and D respectively, with standard error of the mean (SEM) <1% for all three conditions. (F–I) Stimulation of different surface branches leads to isolated constriction. (F) Surface vasculature labeled with Texas Red-dextran. Colored circles indicate size/location of stimulation spots and correspond to time course plots in G, H. For G, total laser power was ~90 mW. For H, total power was ~70 mW. (G, H) Left panels: Difference images showing constriction (blue lines on both sides of vessel) only near stimulation points. Right panels: Time courses of diameter change for the two different stimulations: Error bands are SD across five (G) or four (H) repetitions. Vertical gray band is stimulation interval (100 ms). (I) Population average constriction of vessel segments when targeted (red) or not (blue). N = 8 vessels in 3 animals. Average total power per vessel was 138 mW (range 90–250 mW). Population mean ± SEM from 1 to 5 s following stimulation: Targeted arterioles = 6.5 ± 1%; Untargeted = 0.3 ± 0.4%. (J–M) Stimulation of penetrating arterioles with single-vessel precision. (J) Two penetrating arterioles 200 µm below the cortical surface. Left panels: Difference images when stimulating arteriole #1 (K) or arteriole #2 (L). Each arteriole was stimulated with a total power ~110 mW. Right panels: Time courses of diameter change of the vessels indicated in the left panels. Data presented as mean and SD across seven repetitions. (M) Population average constriction of penetrating arterioles when targeted with stimulation (red) and when not targeted (blue). N = 9 vessels in 3 animals. Average total power applied to each vessel was 130 mW (range 80–190 mW). Population mean ± SEM from 1.5 to 4.5 s following stimulation. Targeted arterioles = 3.8 ± 0.6%; Untargeted = 0.7 ± 0.3%.