Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

The overall goal of this procedure is to document the complete ultra structure of a specific gut sensory cell scattered among other epithelial cells in native intestinal tissue. This is accomplished by first obtaining a 300 by 50 micron tissue segment from the intestine. Next optical Z stacks of the segment Using confocal microscopy are obtained.

Then the confocal data serves as a guide to process and image the tissue using serial block face scanning electron microscopy or SBEM. Finally, the SBEM data is segmented to render the ultra structure of a specific gut sensor cell in 3D. Ultimately correlative confocal microscopy and serial block face scanning.

Electron microscopy are used to show a specific cell in its native tissue and reveal its ultra structure in 3D. This method can help answer key questions in the cell biology field, such as the specific cell to cell interactions that could not be visualized otherwise at a light microscopy level. By using this method, we previously reported a hidden physical chemical relationship between sensory enter endocrin cells and enter ggl.

After isolating the mouse colon, according to the text protocol, place it in nice cold PBS and while submerged cut the tissue open along the mesentary, transfer the tissue into a few drops of PBS on a sheet of dental wax and using a scalpel cuss about six small tissue segments from the distal colon. After preparing 5%low melting aros in PBS, use it to embed the tissue segments in a small plastic container such as the standard size tissue tech cryo mold mount the embedded sections on a vibrating blade microtome and use ice cold PBS to fill the buffer tray. Then cut 300 micron tissue strips at 0.8 amplitude and 0.04 millimeters per second speed.

Reed the 300 micron tissue strips in aros and mount them on a microtone perpendicularly to the blade. Then cut tissue strips at a thickness of 50 microns referring to the text protocol for details before storing blocks in PBS at four degrees Celsius. After imaging tissue blocks using confocal microscopy according to the text protocol to infiltrate and embed tissue sections in resin.

Remove the tissue from PBS and use 0.1 molar co coate buffer to rinse it three times for five minutes each. Once resin has been prepared and tissues have been stained for electron microscopy, according to the text protocol, use it to sandwich tissue blocks between glass slides cured with a liquid release agent after the tissue blocks have been embedded, flat cure the blocks for an additional 48 hours at 60 degrees Celsius. Then pull the slides apart to release the blocks under a dissecting scope, match the orientation of the tissue blocks embedded in resin with that of the confocal micrographs.

To facilitate identifying the regions containing the cells of interest, mount the block flat on a pin containing a conductive epoxy and dry for 30 minutes. Then lay the block flat on a surface and dry overnight at 60 degrees Celsius the following day. Coat the block with colloidal silver liquid.

Maintain the tissue sections flat to ensure slicing of the block at the right angle to facilitate correlating the serial block face and confocal micrographs. After carrying out scanning electron microscopy according to the text protocol, convert the SPEM images from raw DM three format to eight bit TIFF images using a 0.8 Gaussian blur filter in Fiji. Filter the TIFF images.

Scale down the data set to 25%of the original size and save it as a TIFF stack to minimize the amount of RAM memory necessary to handle the data set with the Fiji plugin linear stack alignment with sift in translation mode. Align the stack of SPEM images and use the crop 3D plugin to crop them. To render the data, use the surfaces tool in draw mode and the contour option in draw mode of 50 milliseconds to manually segment and volume render the cell of interest.

Trace contours on every slice of the cell for smoother rendering or every five slices for faster rendering. Use the snapshot tool to export the final images at a resolution of 300 DPI or more using the P-Y-Y-G-F-P mouse. All enteroendocrine cells of the distal part of the small intestine are identified and processed for SBEM as shown here.

A selected tissue segment was imaged by confocal microscopy to obtain Zack images, which are optically spaced one micron apart. Optimizing the dimensions of the tissue blocks is critical for topographical correlation between confocal and SBEM data. This volume rendered view of an enteroendocrine cell in the ileum of the mouse shows a prominent neuro pod extended underneath epithelial cells approximately 60 microns.

By correlating the confocal Z stacks with an SBEM image of the entire block face, the cell of interest is identified here in a block from the distal colon of A-P-Y-Y-G-F-P mouse. It is important to maintain the orientation of the block as flat as possible to match the optical slices in the SBEM images. Once the cell has been identified, SBEM imaging is done as a resolution high enough to identify secretory sles and other cell organelles.

This video contains a rendering in 3D of the enteroendocrine cell. The dataset contains 643 images that span 45 micron of the tissue depth. The cell contains a neuro pod packed with secretory les as well as microvilli.

After watching this video, you should have a good understanding of how to document the complete ultra structure of a specific gut Sensory cell is scatter among other epithelial cells in native intestinal tissue by using fluorescence as a guide to identify the cell or cells of interest and process the tissue for serial block phase scanning electron microscopy.