Церебральный измерения оксигенации крови на основе кислорода зависит от тушения фосфоресценции
Summary
Мы представляем экспериментальные методики измерения парциальное давление кислорода (рО2) в мозговой сосудистой на основе кислорода зависит от тушения фосфоресценции. Подготовка животных и визуализации процедур были определены как для больших поле зрения ПЗС основе изображений рО2 у крыс и 2-фотонного возбуждения основана изображений рО2 у мышей.
Abstract
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.
Protocol
Discussion
Мы продемонстрировали два приложения рО2 измерения в корковых микрососудов на основе кислорода зависит от тушения фосфоресценции. В то время как первый метод, основанный на ПЗС-изображений обеспечивает широкое поле зрения контроля рО2, измерительные парциальное давление кислорода в ?…
Acknowledgements
Мы хотели бы отметить поддержку от американского Национального института здравоохранения грантов R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS067050, R01HL081273 и R01EB007279 и Американской ассоциации сердца грант 0855772D.
Materials
| 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. |
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