A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature and record blood flow dynamics in the intact brain of living rodents is a powerful technique that can be applied to the study of cerebral ischemia or normal brain physiology. Here we will demonstrate how to use two photo microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein. This technique was originally developed by David Kleinfeld in Winfred Dank.

Hi, I'm Ricardo Mota from the Laboratory of Carlos Porter Cayo in the Department of Neurology at the University of California Los Angeles. In a previous video, we showed you the method for implanting a cranial window for chronic imaging experiments in vivo. Today we are going to show you the procedure for imaging blood flow dynamics in the cerebral cortex through a cranial window.

This preparation is very useful because it allows one to image deep inside of the brain using to photon laser microscopy. So let's get it started. In order to image the cerebral vasculature, Animals must be injected intravenously with a fluorescent dye.

We use Dexter conjugated dyes because the dextron moty prevents the dye from crossing the blood-brain barrier and leaking outta the blood vessels. Mice are anesthetized with isof fluorine, 4%for induction and 1.5 to 2%during the injection. Next, the tail is disinfected with three swabs of 70%alcohol with a 26 gauge needle, 75 to a hundred microliters of a 5%solution of R domine Dextran dissolved in saline is injected through the tail vein.

Midway along the shaft of the tail animals may be imaged immediately after the tail vein injection until the die is excreted in the urine, which will turn pink if using ERO domine dye In around two hours following the fluorescent Dexter dye injection, The mouse is anesthetized with isof fluorine. Again, 4%for induction, one to 1.5%for imaging. The mouse is firmly attached using the titanium bar to the microscope stage, which contains a thermal regulated heating pad to keep the animal warm.

Some eye ointment is applied to keep the eyes moist. The cover glass of the cranial window is cleaned with 70%alcohol. The window is positioned parallel to the focal plane and centered in the field of view.

Under the four x objective, it is best to take a photograph of the brain surface vessels with a digital camera. This picture will be used as the image of reference in following imaging sessions to find the image region repeatedly from day to day. The four x objective is then replaced with a water immersion 40 x objective.

Without moving the stage, the digital picture of the view is taken. Again, the coordinates of the stage manipulator are set at zero. We use scan image as the image acquisition software.

This was written in MATLAB by Tom Paul Rudo and Bernardo Sabatini in the laboratory of Carls Voto. We next turn on all the other equipment, the laser, the power meter, the photo multiplier tubes, the amplifiers, et cetera. The area of the window suitable to be imaged is briefly scanned at low magnification to find the best regions.

Once these are identified, the low magnification stack of the chosen region to image is taken and its XY coordinates are annotated to Record blood flow dynamics in small vessels or capillaries. We use line scans along at least 40 microns of the vessel of interest. These single sweeps lasting one second are done using as little laser power as possible, and the coordinates and angle of scanning are written down.

Once the imaging session is over, the animal is moved to a warm chamber where it can recover from the anesthesia. Here we Show a stack of a hundred images taken every five microns with our two photon microscope through a cranial window placed over the somatosensory cortex of a five month old mouse that was injected with r Domine dextran through the tail vein. This sequence of images shows the complex network of large and small blood vessels, including Capillaries.

We have Just shown you a method for visualization of blood flow dynamics in vivo through a cranial window. This is an extremely powerful method for imaging cerebral blood flow dynamics in the cortical vasculature, for example, to investigate alterations in blood flow after stroke. Multiple imaginations can be done at various time in BULs by using repeated tail vein injections of the fluorescent dye.

When doing this procedure, it's important to remember to work in the cleanest and more sterile conditions possible and to have a very steady hand. Okay, and that's it. Thanks for watching and good luck with Your experiment.