In vivo two-photon imaging

Imaging was performed with a Bruker Investigator and a Spectra-Physics Insight X3 laser source. For studies examining arteriolar vasodynamics, chlorprothixene sedation was used. We injected 30 µL of 1 mg/mL chlorprothixene solution intramuscularly (thigh muscle), immediately after cranial window surgery with isoflurane (Sigma C1671). We then injected 2 MDa FITC-dextran (FD2000S; Sigma-Aldrich) retro-orbitally under 2% isoflurane, turned off the isoflurane, and waited 15–30 minutes for chlorprothixene to take effect. FITC-dextran was prepared at a concentration of 5% (w/v) and 0.03 mL was injected. For studies examining capillary diameter, mice were imaged under isoflurane anesthesia (~1.25% MAC in medical air), which leaves the mouse anesthetized but reactive to light toe pinch during imaging. In isoflurane experiments, 2 MDa FITC-dextran was also used for vascular labeling. Body temperature was maintained at 37 °C with a feed-back regulated heat pad. In vivo imaging of FITC-dextran was performed at 800-nm excitation. High-resolution imaging of microvasculature was performed using a 20-X, 1.0 NA water-immersion objective lens (Olympus XLUMPLFLN 20XW). Lateral sampling (x, y) was ~1 µm/pixel (arterioles) or 0.5 µm/pixel (capillaries), and axial sampling (z) was 1 µm/pixel. All lumen diameter quantifications were made in the lateral plane. When imaging with the 20-X objective, laser power ranged between 20–100 mW at the sample, with higher powers required for greater cortical depth. To ablate a single capillary pericyte, a restricted line-scan was applied to only the soma of the target pericyte for ~60 seconds at a power of ~50 mW and excitation wavelength of 725-nm, as previously described (22).