Transmission Electron Microscopy (TEM), fully known as Transmission Electron Microscope, utilizes electromagnetic fields as lenses to focus and direct an accelerated electron beam onto ultra-thin sample sections (typically 70-90nm thick). When electrons interact with atoms in the sample, they undergo scattering at various angles. The scattering angle depends on the sample's density and thickness, thereby generating contrast in the resulting image. This magnified and focused image is then displayed on a detection device. TEM is a high-resolution (0.1nm-0.2nm) microscope capable of extremely high magnification (0.2K-600K×), making it a powerful tool for observing and studying ultrastructural details of materials.

Transmission electron microscopy (TEM) has a wide range of applications in various fields such as cell biology, histology, virology, pathology, molecular biology, and materials science. It can be used to observe the ultrastructure of animal and plant cells, including organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, chloroplasts, vacuoles, and intracellular bacteria, as well as their pathological changes. Additionally, TEM enables the observation of viral particles, exosomes, pathogenic microorganisms, various bacteria and fungi, nanomaterials and nanoparticles, and crystal structures.

I. Basic Principles of Transmission Electron Microscopy

The electron beam emitted from the electron gun travels along the optical axis of the microscope through the condenser lens in a vacuum channel. The condenser lens focuses the beam into a fine, bright, and uniform spot, which illuminates the sample in the specimen chamber. The transmitted electron beam carries structural information from the interior of the sample—fewer electrons pass through denser regions, while more electrons pass through sparser regions. After being focused and primarily magnified by the objective lens, the electron beam enters the subsequent intermediate lens and the first and second projection lenses for comprehensive magnification and imaging. The magnified electron image is then projected onto the fluorescent screen in the observation chamber. The fluorescent screen converts the electron image into a visible light image for observation by the user.

II. Sample Requirements for Transmission Electron Microscopy

2.1 Animal Tissue Samples

① Sampling within 1-3 minutes: Collect tissue samples measuring 2mm × 2mm in size, ensuring they are as thin as possible. If immediate trimming is not feasible, immerse the tissue in TEM fixative for approximately 30 minutes until it hardens before trimming and proceeding with post-fixation. Tissues left beyond this timeframe may not fix properly, compromising subsequent experiments. Strict adherence to this step is critical.

② Precision in sampling: Target the specific region of interest (e.g., renal cortex for glomeruli, pancreatic tail for islets). For tissues prone to curling (e.g., skin, intestines, stomach), adhere them to filter paper before fixation to maintain flatness.

③ Avoid mechanical damage: Use sharp blades to prevent tissue crushing or tearing, and handle samples gently to minimize artifacts from forceps compression.

④ Fixation & storage: Immediately after collection, immerse tissues in TEM fixative for 2 hours at room temperature, then transfer to 4°C for storage and transport. Ensure the fixative does not freeze during storage or shipping. Samples can be stored at 4°C for up to 1 month.

2.2 Plant Tissue Samples

① Sampling requirements are the same as for animal tissues (refer to Section 2.1 for details).

② After placing the tissue in fixative, vacuum infiltration must be performed to ensure the tissue sinks. If vacuum equipment is unavailable, gently press the tissue into the fixative using filter paper to prevent floating. Tissues must not remain on the surface of the fixative, as this will lead to incomplete fixation.

2.3 Cell Samples

Adherent Cells：

Method 1：

① Discard the culture medium from the well-cultured cells and add 2.5% glutaraldehyde fixative at room temperature.

② Fix at room temperature for about 5 minutes, then gently scrape the cells in one direction using a cell scraper (or a small, flat piece cut from a soft rubber cap). Avoid repeated scraping to prevent cell rupture.

③ Use a Pasteur pipette to transfer the cell suspension into a centrifuge tube. Centrifuge at no more than 3000 rpm for about 2 minutes until the cell pellet is approximately the size of a mung bean.

④ After discarding the fixative, add fresh EM (electron microscopy) fixative. Gently lift the cell pellet to suspend it in the fixative.

⑤ Fix at room temperature in the dark for 30 minutes, then transfer to 4°C for storage. Transport with 4°C ice packs, ensuring the fixative does not freeze or form ice during storage and transportation.

Method 2 (for cases where cell morphology is not a critical factor):

① Discard the culture medium from the well-cultured cells and add trypsin.

② After sufficient digestion (avoid over-digestion), neutralize the trypsin with fresh culture medium. Gently pipette to detach the cells, transfer the suspension to a centrifuge tube, and centrifuge at no more than 3000 rpm for about 2 minutes until the cell pellet is approximately the size of a mung bean.

③ Discard the supernatant and add 2.5% glutaraldehyde fixative at room temperature. Gently resuspend the cell pellet in the fixative.

④ Fix at room temperature in the dark for 30 minutes, then transfer to 4°C for storage. Transport with 4°C ice packs, ensuring the fixative does not freeze or form ice during storage and transportation.

For Suspension Cells:Centrifuge the cells to collect a visible pellet (approximately the size of a mung bean). Discard the supernatant and add electron microscopy (EM) fixative. Fix at room temperature for 2 hours, then transfer to 4°C for storage. Transport with 4°C ice packs, ensuring the fixative does not freeze or form ice during storage and transportation.

2.4 Bacterial Samples

For bacteria grown on solid media:Scrape the bacterial colonies along with the agar medium and place them in EM fixative. Fix at room temperature for 2 hours, then transfer to 4°C for storage. Transport with 4°C ice packs, ensuring the fixative does not freeze or form ice during storage and transportation.

For suspended bacterial cells/spores:Centrifuge the bacterial suspension to obtain a visible pellet (approximately the size of a mung bean). Discard the supernatant and add EM fixative. Fix at room temperature for 2 hours, then transfer to 4°C for storage. Transport with 4°C ice packs, ensuring the fixative does not freeze or form ice during storage and transportation.

2.5 Virus

Disrupt the tissue cells, and perform low-speed centrifugation to remove cell debris and collect the supernatant. Ultracentrifugation is then used to isolate the virus (the virus extraction process must be completed by the customer). Suspend the virus in a buffer (e.g., PBS). For long-distance transportation, store and ship at -80°C; for short-distance transportation, store and ship at 4°C. Prepare the negative staining sample as soon as possible and promptly observe and photograph under electron microscopy.

2.6 Exosomes/Vesicles

The customer should complete the extraction and collection of exosomes/vesicles independently. Suspend the sample in a buffer (e.g., PBS) or the preservation solution provided in the kit. For long-distance transportation, store and ship at -80°C; for short-distance transportation, store and ship at 4°C. Prepare the negative staining sample as soon as possible and promptly observe and photograph under electron microscopy. (Since such samples are highly prone to degradation, fresher samples yield better results. It is recommended to complete negative staining and preparation within a few hours; otherwise, the results may be poor or even undetectable.)

2.7 Nanomaterials and Other Inorganic Materials

Prepare the sample directly as powder or suspend it in a buffer (e.g., PBS). The sample can be stored and transported at room temperature (please specify if sonication is required). After negative staining, observe and photograph under electron microscopy.

III. Similarities and Differences Between SEM and TEM

3.1 Similarities：

(1) Both devices use electrons to obtain images of samples.

(2) They share the same main components: an electron gun, electromagnetic lenses, etc.

(3) Both require operation in a vacuum environment.

3.2 Differences：

KMD Bioscience boasts a team of professional scientists and well-equipped imaging laboratories, offering comprehensive Transmission Electron Microscopy (TEM) and Scanning Transmission Electron Microscopy (STEM) services for biological sciences and clinical research, including plant samples, animal specimens, bacteria, and pathological samples. Our seasoned experts provide experimental design guidance for sample collection, preparation, and evaluation. Additionally, we offer professional pathology consultation services for image interpretation and result analysis.

This article is intended for reference by science enthusiasts. It cannot substitute for professional expertise or practical experimental procedures that require more detailed and specialized information. If any content infringes upon rights, please contact the author immediately for removal of the disputed material.