The ultrathin sections of samples provide chemically stabilized internal structures of specimens, generating two-dimensional representations of three-dimensional objects for TEM imaging. Therefore, comprehensive investigation of the three-dimensional ultrastructure of samples is essential. Equally important is recognizing that the production of thin-sectioned specimens results from multiple procedural steps including sample extraction, one or more chemical fixation steps, dehydration and embedding, sectioning, and staining.

Generally, immunoelectron microscopy applications involve direct electron microscopic imaging of antigen-antibody complexes. To enhance microbial detection sensitivity and reveal the ultrastructure of elusive organisms, extensive applications have been developed. When employed for sensitivity enhancement, antibodies can aggregate viruses or bacteria either in solution or on grids for negative staining. Immunogold staining can be combined with negative staining to provide a sensitive technique for identifying and visualizing individual antigens via TEM, which can also be applied to the identification of biological agents themselves.

Materials for processing may include particulate matter such as viral or bacterial suspensions, bacterial or cultured cell samples concentrated into pellets by centrifugation, monolayer cell cultures, or tissue blocks. Due to the volatility and toxicity of fixatives used in the protocol (namely glutaraldehyde, paraformaldehyde, and osmium tetroxide), these substances should be handled with latex or nitrile gloves within a fume hood.

I. Fixation and Initial Processing of Tissue Samples

The critical consideration is the size of the tissue sample, which must be reduced to 1 mm³ or smaller. This critical dimension must not be exceeded to ensure adequate penetration of the fixative during initial processing, post-fixation, bulk staining, and subsequent infiltration steps. The processing steps are as follows:

(1) Obtain a fresh piece of tissue and, as quickly as possible, use a blade to cut the tissue into fragments less than 1 mm thick on a polypropylene cutting board. During the slicing process, it is advisable to cover the tissue with a small amount of TEM primary fixative.

(2) Place several small tissue fragments into a fixation vial containing freshly prepared TEM primary fixative, with a volume at least 20 times that of the tissue pieces.

(3) Incubate the tissue at room temperature for 60 minutes, then transfer it to a 4°C ice bath for another 60 minutes. Alternatively, fixation can be performed overnight at 4°C.

(4) Pour off the fixative from the vial and rinse the tissue multiple times within 30 minutes using a pre-chilled (4°C) phosphate/sucrose wash buffer.

1.2 Post-Fixation Staining

(1) Place the tissue blocks in osmium tetroxide and fix at 4°C for 90 minutes. During fixation, gently agitate the tissue blocks periodically or place the vial on a low-speed platform shaker to enhance osmium tetroxide penetration.

(2) After fixation, decant the solution into a waste container in a fume hood. Rinse the tissue blocks twice with chilled phosphate/sucrose wash buffer, followed by six rinses in cold distilled water to remove residual phosphate buffer.

(3) Bulk-stain the tissues in uranyl acetate staining solution for 90 to 120 minutes at 4°C with periodic agitation. Alternatively, stain in 0.5% aqueous uranyl acetate overnight at 4°C.

(4) Wash the tissues three times in cold distilled water to remove residual uranyl acetate.

1.3 Tissue Dehydration and Embedding

(1) Dehydrate the samples through an ethanol series by sequentially replacing the solution in the wells (10 minutes per step):50% ethanol, at 4°C;75% ethanol, at 4°C;95% ethanol, at room temperature (RT);100% ethanol, at RT

(2) Perform three 10-minute incubations in acetone or propylene oxide, then embed in epoxy resin.

II. Fixation and Initial Processing of Cellular Pellets

This category encompasses samples ranging from cell cultures used for virus isolation and propagation to pure bacterial cultures in growth media. The cell quantity and sample tube type will determine the pellet fixation method and initial processing approach.Various cell culture preparation methods exist for electron microscopy, including scraping monolayers with cell scrapers or generating monodispersed populations through trypsinization. These cells are subsequently concentrated via centrifugation prior to fixation.The pelleted material must be carefully extracted from centrifuge tubes. Depending on the pellet size and stability, it may be processed either as solid tissue or encapsulated in agar to ensure structural integrity during subsequent sample preparation steps.

2.1 Pellet Preparation

Harvest bacterial or cultured cells in buffered saline or serum-free medium, then centrifuge in either rigid-wall or soft-wall microcentrifuge tubes.

2.2 Fixation of Cell Pellets

(1) If the sample is collected in a hard-walled microcentrifuge tube: Carefully remove the supernatant. In a fume hood, slowly add freshly prepared primary TEM fixative, using a volume at least 20 times that of the pellet, and fix at 4°C for 1 hour.

(2) If the sample is collected in a soft-walled microcentrifuge tube: Cut off the tip of the microcentrifuge tube containing the pellet and let the pellet, along with the end of the tube, drop into a fixation vial containing freshly prepared primary TEM fixative. The fixative volume should be at least 20 times that of the pellet, and fixation should be carried out at 4°C for 1 hour.

(3) Replace the fixative with phosphate/sucrose rinse buffer, and carefully remove the pellet from the microcentrifuge tube or the cut tube tip.

2.3 Embedding Cells in Agar and Refixing

(1) If the thickness of the pellet is less than 1 mm: Carefully transfer the pellet into 2.5% molten agar (maintained at 40°C–60°C) deposited on a sealing film, and keep it suspended in the agar until solidification.

(2) If the thickness of the pellet is greater than 1 mm: Cut the pellet into 1 mm-thick slices from top to bottom. To preserve the integrity of the pellet, gently transfer the sliced sections into droplets of molten agar deposited on sealing film, suspending them in the agar until solidification.

(3) Remove excess solidified agar using a cell scraper to enhance the exchange of fixatives and solvents.

(4) Transfer the pellet or slices into a fixation vial containing fresh fixative and incubate at 4°C for an additional 90 minutes.

(5) After fixation, decant the fixative from the pellet and replace it with phosphate/sucrose protease buffer at 4°C. Incubate for 10 minutes at 4°C, then replace with fresh phosphate/sucrose protease buffer and incubate for another 10 minutes at 4°C.

2.4 Post-Fixation Staining (Same as Step 1.2)

KMD Bioscience has a team of professional scientists and a well-equipped imaging laboratory, offering comprehensive Transmission Electron Microscopy (TEM) and Scanning Transmission Electron Microscopy (STEM) services for biological and clinical research, including plant samples, animal samples, bacteria, and pathological specimens. Our experienced experts provide guidance on experimental design for sample collection, preparation, and evaluation. Additionally, we offer professional pathological consultation on imaging and results.