This article details a method for examining mouse brain tissue, specifically the hippocampus, using serial block-face scanning electron microscopy (SBFSEM). The technique allows for high-resolution imaging and 3D reconstruction of mitochondrial structures within neuronal compartments.
Take a trimmed, resin-embedded mouse brain tissue section containing the hippocampus.
The section is stained with heavy metals to enhance contrast under an electron microscope.
Mount the section onto an aluminum pin using cyanoacrylate glue.
Coat the block sides with colloidal silver paste to create a conductive path to the aluminum pin.
Examine the tissue block using a serial block-face scanning electron microscope (SBFSEM).
Within the SBFSEM, the ultramicrotome sequentially slices the block into ultrathin layers while a low-voltage electron beam scans the exposed surface.
A backscattered electron detector captures high-resolution 2D images, revealing mitochondria and surrounding cellular structures.
Using appropriate software, adjust the brightness and contrast to improve image clarity.
Align the sequential images to reconstruct a 3D volume of the brain tissue. This 3D reconstruction allows precise mapping of mitochondrial morphology, spatial distribution, and their relationships within neuronal compartments.
Trim the samples to the area of interest and mount them onto an aluminum pin using gelling cyanoacrylate superglue. Then coat the sample with colloidal silver paste around the sides of the block to provide a conductive path to the aluminum pin. Examine the tissue specimens using a scanning electron microscope system equipped with an in-chamber ultramicrotome stage and a low kilovolt backscattered electron detector.
Image the samples with these settings. Begin analysis by selecting image type, then 8-bit to convert the images to 8-bit TIFF format from the original proprietary 16-bit. If automatic contrast and/or brightness conversion during this step is not ideal for images, reopen the 16-bit images and press image. Adjust brightness contrast.
Select a range that works for all images and press Apply. Then perform the conversion. If there was unacceptable image movement between slices, align the image stacks by selecting Plug-ins, Registration and linear stack alignment with SIFT and set the alignment for translation only mode rather than rigid body.
Enlarge the Canvas size prior to registration by selecting image adjust Canvas size. Alternatively, reduce the image to an area of interest by selecting the desired region and then cropping it. If necessary, scale images to a smaller, more manageable size using image j, then selecting image and scale.
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