Three-dimensional Imaging of Immunolabeled Astrocytes Using Confocal Microscopy

Take an immunostained mouse brain section with fluorescently labeled astrocytes. The astrocyte's cytoplasm fluoresces red, while the nucleus fluoresces blue.

Place the slide on a confocal microscope's stage under a suitable objective lens.

Using the acquisition software, adjust the focus and select the fluorophores labeling the astrocyte's cytoplasm and the nucleus.

Begin live imaging and optimize imaging parameters to reduce noise in the acquired image.

Under acquisition parameters, select a low-speed scanning mode for capturing a higher-quality two-dimensional image.

Identify the uppermost position of the astrocyte as the starting point for the Z-stack and the lowest position as the endpoint.

Define the number of slices covering the full cell depth.

Merge the slices to generate a three-dimensional image of the astrocyte.

To begin confocal microscopy, place a slide onto the microscope stage and select the 63x objective. After opening the Acquisition software, click on Smart Setup in the Acquisition tab and select the proper fluorophores. Here, DAPI and Alexa Fluor 555 are used.

Next, click on Best signal, followed by Apply. Then click on Set Exposure to allow the computer to determine the optimal exposure parameters.

To optimize the image, open the Channels window and switch the image to Live. Adjust the focus if needed. Then click on 1 AU to optimize the pinhole. Adjust gain for best intensity.

Finally, set the digital gain between 2 and 3. After this optimization, stop the live image and open the Acquisition Mode window. Adjust the frame size by clicking on X by Y, and select 1024 by 1024. Also, choose a slow speed.

Next, open the Averaging tab and select a number greater than or equal to 4. Then click the Snap button and save the captured 2D image. To set up 3D imaging, open the Smart Setup tab and mark the Z-Stack item, which will cause the stack window to open.

To set the first and last positions for the Z-Stack, again, click Live and adjust the focus to the uppermost position of the astrocyte. Click on Set First. Now adjust the focus to the lowest position of the astrocyte and click on Set Last. Then stop the live view.

Next, choose the interval by clicking on Optimal to set the number of slices. Here, the interval is 1.01 micrometers. Finally, click on Start Experiment and save the images once the scan is complete.