TEM Techniques

This is an example of ImmunoSorbant Electron Microscopy (ISEM), which I used throughout my graduate work to verify the presence, antigenicity, and structural integrity of my virus purifications. A major advantage of the technique is the ability to adsorb the antigen (virus) to the grid while reducing nonspecific debris, commonly seen in negatively stained preparations.

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TEM thin section for morphological study: mouse

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This is just one example of the many mammalian tissues that I've worked with over the years. The large cell seen in the center of both images is a Kupffer cell in a transgenic mouse liver. Vibrotome sections of liver were embedded such that they could easily be viewed through a light microscope after polymerization. Thin sections of the selected region were cut en face, and serial sections were picked up on 50 mesh grids to allow for these 2,500x shots.

Some of the tissue samples that I have more experience with include: white and brown adipose tissues, myocardial tissues, Dorsal root and ciliary ganglia, Liver, lung, aorta, iris, hardarian gland, and a variety of tissue culture cells.

This image shows the three major filtration components of the kidney. The top peg-like structures are sections through a single fenestrated epithelial cell called a podocyte. Under this is the basement membrane of the glomerulus, followed by a fenestrated endothelial cell. The dark object in the lower left corner is part of a red blood cell.

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TEM thin section for morphological study: Aphid

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My graduate work involved tracking virus through the four cells of the accessory salivary gland in aphids. Because of the small size of my target tissue, aphids were embedded cut in half, cuticle intact. Typically such samples lead to infiltration problems, and separation of the resin from the chitinous cuticle. This potential problem was avoided by using a combination of long infiltration steps, and Spurr's resin. With the protocol I developed, I did not need to depend on support films, such as Formvar, to keep my samples intact.

All three of the images above are of aphid accessory salivary gland. In the image on the far left, a cross section of tracheole wall can be seen as convoluted material on the right-hand side of the image. These chitinous branches of the gas exchange system in insects extend to all cells of the organism.

The second image features an unidentified structure that was common in the ASG's. Note that this structure lacks a membrane, and is composed of a cluster of small repeating units. This structure is typically seen in a field of rough ER, as show here.

The third image is a film micrograph of the accessory salivary gland in the aphid S. avenae. The large opening is a tracheole, while the dark structure on the bottom left is a lysosome. The basal lamina of this ASG is impregnated with Barley Yellow Dwarf Virus, which can be seen in the larger versions (try links).

TEM of cultured cells containing retrovirus

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The infected cells shown above are an immortalized line of lymphocytes derived from a human leukemia patient. Although Epstein-Barr virus is known to replicate in cultured lymphocytes, this is not the virus seen here. The above images show classic budding morphology of retrovirus.
Note that the virus particles form at the plasma membrane. Each particle has a dense staining core, and a tight lipid envelope. In addition, the particles are 90 nm wide, placing them into the virus family retroviridae. (see: The International committee on Taxonomy of Virus (ICTV) web site for more information)

Compare SEM and TEM images

TEM Immuno-labeling LR White en face sections

The figure to the left shows specific actin labeling in primary chicken embryonic fibroblast cells. The samples were brought to the AIF to develope an immuno-gold labeling protocol, and actin was used as a positive control.

The CEF cells monolayers were embedded in LR White and cut en face. This allowed a large cytoplasmic area to be examined without having to disturb the cells by scrapping. Preliminary experiments showed scapping to result in unexceptable levels of mechanical damage.

Careful examination of the full screen version of this image shows gold label in the upper image, and vertially no gold label in the control image.

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Light fixation and embedding in LR White, an acrylic resin, has it's drawbacks, but preservation of cytoskeletal components does not have to be one of them. This image shows fine cell processes approximately 80-90nm in diameter. TEM sections are typically in the range of 90 nm, signifying the fine processes you see in the image are as deep into the plastic as they are wide.

Membranes are not seen in these images because osmium tetroxide was not used.

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