We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.
Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer’s disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range.
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.
1. Wide field of view imaging of pO2 in cortical vasculature of a rat
2. High resolution two-photon pO2 imaging in cortical micro vasculature of a mouse
3. Representative Results:
Figure 1. Panel (a) on the left side of this figure displays a wide field of view image of oxygen pressure before the arrival of a CSD wave. Panel (b) on the right side shows the temporal evolution of the average oxygen pressure during CSD propagation within the region of interest marked on panel (a).
Figure 2 (avi movie): This movie shows the temporal evolution of the oxygen pressure in the whole cranial window during propagation of the CSD wave. Scale bar indicates oxygen pressure in millimeters of mercury.
Figure 3. 3D projection of the imaged vasculature stack. The shades of grey represent a volumetric vessel mask, created based on the structural image.
Measured pO2 values are color-coded. Scale bar is 200 micrometers. Reprinted with permission from Nature Publishing Group.7
We demonstrated two applications of the pO2 measurement in cortical microvasculature based on oxygen-dependent quenching of phosphorescence. While the first method based on CCD imaging provides wide field of view monitoring of pO2, measuring partial pressure of oxygen in cortical microvasculature based on 2-photon microscopy provides capillary resolution and allows imaging in depth. Both methods provide high speed and high signal-to-noise measurements. In addition, the phosphorescence lifetime measurement of pO2 is largely insensitive to the changes in the optical parameters of the tissue during the experiment, which is usually a concern for the other optical imaging techniques that have a contrast mechanism based on intensity. Presented instruments allow quantitative analysis of the dynamic delivery of oxygen and the brain tissue metabolism that will lead to a better understanding of neurovascular coupling in normal and diseased brain
We would like to acknowledge support from US National Institutes of Health grants R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS067050, R01HL081273 and R01EB007279 and American Heart Association grant 0855772D.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Glycopyrrolate | Reagent | American Regent Inc. | NDC 0517-4605-25 | Used to control pharyngeal, tracheal, and bronchial secretions. |
Lidocaine HCL | Reagent | Hospira Inc. | NDC 0409-4277-01 | Used as the local anesthesia during surgeries. |
Isoflurane | Reagent | Baxter Healthcare Corp. | NDC 10019-360-40 | Used as a general inhalation anesthetic drug during surgeries and as a general anesthesia during experiments with mice. |
Alpha Chloralose | Reagent | Sigma | C0128 | Used as a general anesthesia during experiments with rats. |
Fluorescein isothio-cyanate–dextran | Reagent | Sigma | FD2000S | Administrated to create ~ 500 nM concentration in blood. |