[Figure 2].

Experimental setup and imaging protocols. (a) Schematic of our home-built multimodal optical system featuring primary components of the system. A 50 kHz spectral-domain OCT system was designed to partially share the imaging optics with the two-photon microscope. A Hg:Xe arc lamp in combination with a CCD camera was used for OISI. EOM: electro-optic modulator, SH: shutter, GM: galvanometer mirror pair. (b) A CCD image of brain surface vasculature in the mouse barrel cortex showing the ROIs where various optical measurements were performed (red ROI: capillary RBC flux, pO₂, and microvasculature imaging, blue ROI: Doppler OCT imaging, and green ROI: OCT intensity imaging). (c) A representative OCT intensity B-scan image extracted from a volumetric OCT image. White arrows indicate the boundary between the gray matter (GM) and the corpus callosum (CC), which appears as a bright band in the image. (d) Survey scan images of cerebral microvasculature of the region outlined with the red square in (b) obtained by two-photon microscope at two imaging depths (z=0.4 and 0.9 mm). Two representative fluorescent intensity time courses acquired within the capillaries at the locations indicated by the red dots in the survey angiograms are presented on the right. (e) A 3D angiogram of the mouse cortex acquired by the two-photon microscope at the location outlined by the red square in (b). One representative 2D plane from the angiogram acquired at a depth of 200 μm showing pO₂ measurements from different capillary segments. pO₂ values were color-coded (in mmHg) and spatially co-registered with the angiogram. (f) A 3D Doppler OCT image showing an axial velocity map of the diving vessels at the location outlined by the blue square in (b). (g) An OIS image of the cranial window obtained by calculating the relative intensity difference between the post-stimulus response image and pre-stimulus baseline. The region of activation is manually selected from the OIS image as indicated by a black square. The lower panel shows a time course of the relative intensity change due to sensory-evoked hemodynamic response induced by a 2-s-long whisker stimulation, averaged over the selected region of interest. Scale bars: 400 μm for (b) and (c), 100 μm for (d), (e), and (f), and 500 μm for (g).