This article describes a method for imaging cortical blood flow and oxygenation in an anesthetized rat using spectral data collection. The technique involves capturing diffuse reflectance from cortical tissue to analyze its structure and function.
Begin with an anesthetized rat prepared with a cranial window exposing cortical blood vessels and tissue.
Place the rat on an imaging stage, ensuring it remains connected to a mouthpiece delivering anesthesia and a gas mixture to regulate oxygen levels.
Focus the camera on the brain surface. Pass broadband light through a filter to isolate specific wavelengths.
A light guide and collecting lens direct the filtered light onto the cortical tissue.
Within the tissue, light is absorbed by hemoglobin, depending on its oxygenation state, and scattered by cells, producing diffuse reflectance.
Capture this scattered light using the camera, optimizing brightness by adjusting integration time.
Repeat imaging across multiple wavelengths with a rotating filter wheel to collect spectral data.
Finally, block the light path to capture a dark image, then subtract it from spectral images to remove noise.
These processed images reveal cerebral blood flow, oxygenation, and tissue structure.
Begin by gently placing the rat on the stage, and slowly adjust the stage level so that the camera can focus on the surface of the rat brain. Within the image acquisition software, select the Save command from the File menu to save an image to a file, naming it according to the sample and wavelength.
Then change the filter location by rotating the filter wheel. Repeat the image acquisition at each of the nine wavelengths. Next, turn off the halogen lamp light source. Block the light path to the monochromatic charge-coupled device camera system using a shielding plate. Finally, select the Save command from the File menu, and choose a file name that identifies the sample.
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