PMC

RBC: red blood cell, IOS: intrinsic optical signal, NADH: nicotinamide adenine dinucleotide, O₂: oxygen.

A simplified IOS imaging set up was configured from a dissecting scope with a camera port, a CMOS camera, and a 2 × 3 LED array angled to illuminate the imaging area (). An anesthetized mouse with a cranial window sits in a steriotaxic frame below the scope for imaging. The light reflected by the brain surface is collected through the scope by the camera after passing through an emission filter to select the reflected light from the LEDs. Inset: In dissecting scopes without a dedicated filter holder, a 15–18 mm diameter emission filter is placed in the space between the camera port (tube) lens, and the camera adaptor.

(Steps 1–17) In an anesthetized mouse, the skull over the hind limb somatosensory cortex is exposed (Left). A window is cut through the skull and carefully removed to expose the brain for imaging (red circle on left corresponds to the image on right). A glass coverslip window is then sealed to the brain using low melting temperature agarose (Right). A well formed of cyanoacrilate glue helps keep the agarose in place as it cools and solidifies. Notice the window is clear of blood, and all large surface vessels are intact. Approximate locations of midline and coronal sutures (intersecting at bregma) are shown. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California and the Massachusetts General Hospital Subcommittee on Research Animal Care with National Institutes of Health guidelines.

(Steps 18A) (a) Pseudocolored red IOS signal changes in response to hind limb mechanical vibration stimulus starting at 0 s (300 ms duration). The IOS image data set is an average from 10 individual stimulus trials. IOS Images were normalized to their average pre-stimulus basal values (set to 0) for each pixel, and expressed as percent change in reflected intensity (I) from baseline (ΔI/I_o,%). Images were smoothed with a 3D filter. Boxes indicate region of interest (ROI) for panel b, placed to avoid any large surface vessels. Scale bar is 0.5 mm. (b) Red IOS time course of the parenchymal ROI in (a) in response to stimulus. Curve is an average of 10 trials, and shaded area indicates standard deviation. A dashed line denotes the stimulus start. Red IOS signals are typically multiphasic in vivo with a negative “initial dip,” followed by a positive “overshoot” and second dip or “undershoot.” The overshoot indicates deoxyhemoglobin washout and is a measure of oxygen delivery to the active brain region. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines.

() (a) Large field of view image of the pial vasculature inside the cranial window taken by a charge-couple device camera under green-light illumination is used to identify imaging locations. (b) A two-photon image of FITC-dextran (2000 kDa) labeled blood plasma 100 μm below the cortical surface at the location marked with the red square in a identifies the vascular structure at the region and depth selected for pO₂ imaging. (c) Phosphorescence intensity survey image at the same location as b illustrating regional phosphorescence probe labeling. Black dots represent locations pre-selected for phosphorescence lifetime measurements. (d) Phosphorescence lifetime measurement from one of the grid point locations. 2000 decays were combined to generate the curve. Shaded area indicates 10 μs excitation. (e) Measured pO₂ values overlaid on the microvascular structural image presented in the panel b. pO₂ values (in mmHg) are color coded. (f) Histogram analysis of the pO₂ values in (e). (g) Microvascular angiogram at the location marked by the red square in a. A 300 μm-thick microvascular image stack obtained at the end of the experiment by two-photon imaging of the blood plasma relabeled with Rhodamine B-dextran (70 kDa) robustly labels and identifies the vasculature present in the vicinity of the pO₂ measurements, providing a vascular map of the region. (h) Computer-rendered projection of the segmented three-dimensional microvascular image stack from g with morphologically identified arterioles, capillaries, and venules falsely colored in red, green, and blue, respectively. Scale bars: 200 μm. This procedure was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care with National Institutes of Health guidelines.

(Step 18C) (a) Representative two-photon images of SR101 (red) and NADH (blue) fluorescence were acquired in the somatosensory cortex, 50 μm from the brain surface. SR101 and NADH images were recorded over time before and in response to an electrical hind limb stimulus (10 s duration at 10 Hz, 2 ms pulses). Representative images before (baseline) and at the time of the NADH peak fluorescence change are shown. NADH is intrinsically fluorescent, where increased fluorescence intensity typically correlates with the decreased cortical tissue oxygenation. SR101, a dye that labels astrocytes and oligodendrocytes, is added to the brain tissue and used as a non-functional control marker to correct for hemodynamic changes during stimulus. Scale bar is 20 μm. (b) Time course of NADH fluorescence before, and after an electrical hind limb stimulus starting at0 s. NADH fluorescence plotted over time is corrected for blood flow changes using the SR101 images as previously described. Dashed line indicates stimulus start. The example shows data from one animal. For comparison, the corrected NADH signal time course from a control littermate animal is plotted. The response in the control animal has a much smaller amplitude compared to Pdgfrβ^+/− mouse, likely because the control animal better tolerates metabolic demand of the relatively short stimulus. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines.

(Step 18A) (a) The absorption spectra of oxy- and deoxyhemoglobin, expressed as the molar extinction coefficient. Larger values indicate greater absorption. Dashed lines indicate wavelengths used for green (530 nm) and red (630 nm) IOS imaging. (b) Image of the surface of the somatosensory cortex through a cranial window during IOS imaging (far left), and pseudocolored IOS signal changes in the same area in response to hind limb electrical stimulus starting at 0 s (10 s duration at 10 Hz, 2 ms pulses). The IOS image data set is an average from 10 individual stimulus trials. IOS Images were normalized to their average pre-stimulus baseline values (set to 0) for each pixel, and expressed as percent change in reflected intensity (I) from baseline (ΔI/I_o,%). Images were smoothed with a 3D (x, y, time) spatial filter. Boxes indicate region of interest (ROI) for panel b, placed to avoid any large surface vessels. Scale bar is 0.5 mm. (c) Green IOS time course of the parenchymal ROI in (b) in response to stimulus. Curve is an average of 10 trials, and shaded area indicates standard deviation. A dashed line denotes the stimulus start. Green IOS signals are typically monophasic in vivo with a negative deflection indicating increased hemoglobin absorbance due to increase in blood flow. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines. Panel a data compiled by Dr. Scott Prahl, Oregon Institute of Technology (http://omlc.org/spectra/hemoglobin/index.html).

(Step 18B) (a) An individual FITC-dextran (70 kDa) dye-labeled capillary is shown with a linescan location perpendicular to the capillary marked for diameter measurement (scale bar is 5 μm). (b) High resolution linescans perpendicular to the capillary are acquired over time before, and in response to an electrical hind limb stimulus starting at 0 s (10 s duration at 10 Hz, 2 ms pulses) using a two-photon microscope. The linescans are built up over time into a linescan image or kymograph (scale bar is 2.5 μm). (c) The kymograph image then undergoes pre-threshold filtering with a 2D Gaussian filter. Then the image is thresholded to separate the vascular signal from the background (see ), yielding a binary black and white image from which the capillary diameter is determined for each line to create a time course trace of capillary diameter changes in response to stimulus. The diameter time course trace is normalized to its basal value (see ), and diameter changes are expressed as a percentage of basal diameter (set to 0), resulting in the “raw” capillary diameter time course trace. A dashed line denotes the stimulus start. (d) The raw time course data is subsequently subjected to low-pass, notch, and box filters to reduce noise and breathing artifacts, resulting in the final “Filtered” capillary diameter time course trace in response to stimulus, as a percentage of basal diameter (see ). (e) To determine the time to reach 50% peak dilation, a sigmoid curve is fit to the diameter dilation time course. The time at which the sigmoid curve reaches 50% maximal value corresponds to the capillary time to 50% peak dilation, and is marked with a gray dot and dropline to the time axis. Arrowhead indicates time to 50% peak dilation for this vessel. (f) The measurement process and sigmoid fit is repeated for all diameter time course traces derived from individual capillary responses to stimulus recorded in a single animal. Sigmoid fits were normalized between basal diameter (set to 0) and maximal diameter change in response to stimulus (set to 1). The average sigmoid curve fit (thick red line) were derived from the individual sigmoid fits from multiple capillaries from the same wild type mouse. A horizontal dashed line at 0.5 indicates 50% of maximal value, and arrowheads below the time axis indicate times when individual and mouse average capillary sigmoid fits reach 50% maximal diameter. The same process can be used to measure diameter changes in response to stimulus in arteriole vessels. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines.

(Step 18B) (a) An individual FITC-dextran (70 kDa) dye-labeled capillary is shown with a linescan location along the center of the capillary marked for RBC velocity measurement (scale bar is 5 μm). (b) High speed linescans of the capillary are acquired using a two-photon microscope. The linescans are built up over time into a linescan image or RBC kymograph (scale bar is 5 μm). Dark stripes indicate the passage of RBCs through the vessel. The angle of the stripes indicate the speed at which the RBCs are traveling, and are used to measure RBC velocity. (c) Velocity information is extracted from the kymograph using a Matlab routine developed by Kim, et al in response to an electrical hind limb stimulus starting at 0 s (10 s duration at 10 Hz, 2 ms pulses). A dashed line denotes the stimulus start. The resulting “Raw” capillary RBC velocity time course trace typically exhibits a strong heartbeat artifact. Inset: Enlarged view of a portion of velocity trace indicated by the bracket illustrating a heartbeat artifact of similar magnitude as reported by^,. (d) The raw capillary RBC velocity trace is subsequently normalized to its baseline velocity, and RBC velocity changes are expressed as a percentage of basal baseline velocity (set to 0). The RBC velocity time course traces is passed through low-pass, notch, and box filters to reduce noise, heartbeat, and breathing artifacts, resulting in the final “Filtered” capillary RBC velocity trace (see text for details). (e) To determine the time to reach 50% peak velocity, a sigmoid curve is fit to the RBC velocity increase time course, similar to diameter measurements. The capillary time to 50% peak velocity corresponds to the sigmoid 50% peak point, and is marked with a gray dot and dropline to the time axis. Arrowhead indicates time to 50% peak RBC velocity for this capillary. (f) The measurement process and sigmoid fit is repeated for all capillary RBC velocity data recorded in an individual animal. Sigmoid fits were normalized between basal RBC velocity (set to 0) and maximal velocity change in response to stimulus (set to 1). The average sigmoid curve fit (thick red line) was derived from the individual sigmoid fits from multiple capillaries from the same wild type mouse. A horizontal dashed line at 0.5 indicates 50% of maximal value, and arrowheads below the time axis indicate time individual and average capillary sigmoid fits reach 50% maximal velocity. The same process is used to measure RBC velocity changes in response to stimulus in arteriole vessels. This procedure was approved by the Institutional Animal Care and Use Committee at the University of Southern California with National Institutes of Health guidelines.