TEM/IEM Sample Preparation

The ultra-thin slice of the sample provides a chemically stable internal structure of the sample, which is used to provide a two-dimensional view of the various parts of the image representing a three-dimensional object in TEM. Therefore, the three-dimensional ultrastructure of the sample must be studied in depth. Equally important, the emergence of thin sliced samples results from multiple steps in the protocol, including extraction of the sample, one or more chemical fixation steps, dehydration and embedding, slicing, and staining.

In general, the application of immunoelectron microscopy involves direct electron microscopy imaging of antigen-antibody complexes. To improve the sensitivity of microbial detection and reveal the ultrastructure of elusive organisms, a wide range of applications have been developed. When used to increase detection sensitivity, antibodies can concentrate viruses or bacteria on a solution or grid for a negative stain. Immunogold staining can be combined with negative staining to provide a sensitive technique for identifying and visualizing individual antigens via transmission electron microscopy, and can be used for the identification of biological agents themselves.

The material to be treated may be particles, such as viral or bacterial suspensions, bacterial or cultured cell samples concentrated into particles by centrifugation, monolayer cell cultures, tissue blocks. Due to the volatility and toxicity of the fixatives used in the program (glutaraldehyde, paraformaldehyde and osmium tetroxide), these items should be treated with latex or nitrile in the fume chest.

1.Tissue sample processing

1.1 Fixation and initial treatment of tissue samples

The key consideration is the size of the tissue sample, which must be reduced to 1mm3 or smaller. This critical size must not be exceeded to ensure adequate penetration of the fixative during initial machining, post-fixing, integral dyeing and subsequent penetration steps. The procedure is as follows:

(1) Obtain a piece of fresh tissue and cut the tissue into pieces less than 1 mm thick on a polypropylene cutting board with a blade as soon as possible. During sectioning, it is best to cover the tissue with a small amount of TEM primary fixative.

(2) Place several small pieces of tissue into a stationary vial containing the newly prepared TEM primary fixative, the volume of which is at least 20 times the volume of the tissue block.

(3) The tissue was cultured at room temperature for 60 minutes and then moved to a 4℃ ice bath for 60 minutes. Or keep it at 4℃ overnight.

(4) Pour out the fixing solution from the vial, rinse the buffer with fresh phosphate/sucrose pre-cooled at 4°C, and clean the tissue several times within 30 minutes.

1.2 Post-fixation staining

(1) The tissue blocks were placed in osmium tetroxide and fixed at 4℃ for 90 minutes. After fixation, the tissue block is often shaken, or the vial is placed on a platform shaking table at a low speed to promote the penetration of osmium tetroxide into the tissue.

(2) After fixing, pour the solution into the waste container in the fume hood. The tissue blocks were rinsed twice in a frozen phosphate/sucrose rinse buffer, then six times in cold distilled water to remove the phosphate buffer.

(3) The whole stained tissue was placed in uranyl acetate staining solution for 90 to 120 minutes and periodically stirred at 4℃. Or stain in 0.5% uranyl acetate solution and leave overnight at 4°C.

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

1.3 Dehydrated and embedded tissue

(1) Through the ethanol series dehydration, the solution in the hole was replaced with the following solution successively, for 10 minutes each time: 50% ethanol, 4℃; 75% ethanol, 4℃; 95% ethanol, room temperature; 100% ethanol, room temperature.

(2) Soak in acetone or propylene oxide three times, each time for 10 minutes, embedded in the epoxy resin.

2. Fixation and initial processing of cell particles

The samples range from cell cultures used to isolate and multiply viruses to pure bacterial cultures in growth media. The number of cells and the type of sample tube will determine the type of particle to be fixed and the initial sample treatment will be performed. Cell culture methods for electron microscopy range from scraping monolayers with cell scrapers to producing monodisperse populations by trypsinization. These cells are concentrated and then fixed by centrifugation. The granular material must be removed from the centrifuge tube and, depending on the size and stability of the particle, it can be treated as a solid tissue or it can be encased in AGAR to ensure integrity in subsequent steps of the sample preparation process.

2.1 Capture particle

The bacteria or cultured cells were harvested in buffered saline or serum-free medium and centrifuged in hard-wall or soft-wall microcentrifuge tubes.

2.2 Fixed cell

(1) If the sample was collected in a hard-walled microcentrifuge tube: carefully remove the supernatant. In the fume hood, the newly prepared TEM primary fixative, whose volume is at least 20 times the particle volume, is slowly added and fixed at 4°C for 1 hour.

(2) If the sample is collected in a soft-wall microcentrifuge tube: cut the tip of the microcentrifuge tube containing the sample particles and drop the particles together with the end of the tube into a fixing bottle containing the newly prepared TEM primary fixative, which is at least 20 times the volume of the particles, and fix it at 4°C for 1 hour.

(3) Replace the fixative with phosphate/sucrose flush buffer and carefully remove the particles from the microcentrifuge tube or cutting tip.

2.3 The cells were encased in AGAR and fixed again

(1) If the thickness of the particles is less than 1mm: carefully transfer the particles to 2.5% molten AGAR (kept at 40°C -60°C) to deposit on the sealing film and remain suspended in the AGAR until solidified.

(2) If the thickness of the particle is greater than 1 mm: Cut the particle from top to bottom into 1 mm thick slices. To maintain the integrity of the particles, the particle slices are carefully transferred to a molten AGAR drop deposited on the sealing film, and the slices are suspended in the AGAR until it solidifies.

(3) Excess cured AGAR is removed with a cell scraper to enhance the exchange of fixatives and solvents. (4) Transfer the particles or slices to a fixed bottle containing fresh fixing solution and incubate at 4°C for another 90 minutes.

(5) After fixation, pour out the fixing solution from the precipitation and replace it with phosphate/sucrase buffer at 4°C. Incubate at 4°C for 10 minutes, then replace with fresh phosphate/sucrase buffer and incubate at 4 ° C for another 10 minutes.

2.4 Integral dyeing after fixation (same as step 1.2)

With specialist scientists and imaging laboratories, KMD Bioscience offers a full range of transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) services for bioscience and clinical research including plant samples, animal samples, bacteria and pathological specimens. Our experienced experts can provide experimental design guidance for sample collection, preparation and evaluation. We also offer professional pathology consultations on images and results.