This article describes a method for imaging the blood-brain barrier using confocal microscopy. It details the preparation of mouse brain sections and the settings required for optimal imaging.
Begin with a confocal microscope software and set the image acquisition parameters.
Then, take a mouse brain section mounted on a glass slide. This section is stained with antibodies targeting blood-brain barrier components.
The blood-brain barrier is a selective cellular interface between the bloodstream and the brain to maintain homeostasis.
Apply a drop of immersion oil with a desired refractive index onto the coverslip to optimize resolution and minimize light refraction.
Place the slide on the microscope stage and use the epifluorescence lamp to visualize the sample.
Using the appropriate filters, locate the DAPI-stained nuclei and identify the capillary segment for imaging.
Start the live scanning and adjust the parameters for each fluorescent channel to capture high-quality images.
Finally, define the start and end points for Z-stack acquisition to generate a three-dimensional image of the blood-brain barrier.
In the software's Acquisition menu that controls the microscope, set the image acquisition parameters. In the dropdown menu of image size, select a value of 1,024 by 1,024 pixels. Modify the pixel size to a value between 200 and 300 nanometers by adjusting the value of the Zoom menu.
In the acquisition Speed dropdown menu, select a value of 400 hertz. Next, in the System Settings menu, select the pixel depth, and change the value to 12-bit. Then, in the microscope software, within the Acquisition tab, toggle on the option for sequential frame acquisition. Set the optical section thickness to a value between 0.75 and 1 micrometer for each channel, by modifying the value in the Pinhole menu.
Add a drop of immersion oil with a refractive index of 1.52 on top of the coverslip, with the brain section to match the refractive index of the glass coverslip and objective. Subsequently, place the sample in the microscope, and turn on the epifluorescent lamp to visualize the sample using the microscope binoculars. Next, press the button to start the live scan mode. During scanning, adjust the gain and laser intensity for each channel to maximize the dynamic range of the images and avoid pixel saturation. Afterward establish the begin and end sections for performing optical Z-stacks that span the whole volume of the capillary, and use a step size of 0.45 micrometers.
Importantly, all acquisition settings should be kept identical during the imaging session.
繁體中文
