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The light digital microscope can be used to visualize structures as small as ~1 micron

Transmission electron microscopy (TEM)

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Introduction

Transmission electron microscopy (TEM) is an important tool in the expanding field of biomedical research and microbiology. The high resolution provided by the electron beams accelerated at high voltage makes the TEM indispensable for detailed analysis.

The specimen is an ultrathin section or a suspension on a grid. The electron beam is passed through the specimen. An image is formed by the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image of the sample is then magnified and focused onto an imaging device.

Transmission electron microscopy is a major analytical technique used in the physical, chemical, and biological sciences. Transmission electron microscopy has a wide range of applications in cancer research, virology, materials science, nanotechnology, and semiconductor research.

Principle

The transmission electron microscope works like a slide projector. The electron source generates a beam of accelerated electrons which transmits through the specimen.

The transmitted beam replicates the reflected and scattered electron and forms an enlarged image of the specimen on the screen. The transmission of the electron beam is dependent on the properties such as density, composition, etc. of the sample or material being examined.

Apparatus

The basic part of the transmission electron microscope is the electron source. It is a V-shaped filament made of tungsten wreathed with an electrode. The electrode emits the electron beam from a small area of the filament.

The electron beam is then focused in a small, thin, and coherent beam by a pair of condenser lenses. The first lens determines the size range of the final spot that strikes the specimen. The second lens changes the size of the spot on the sample.

The condenser aperture restricts the electron beam and filters out the unwanted scattered electrons. The scattered and reflected electrons are then focused on an image by the objective lens. Then the projector lens expands the beam on the detector screen. The screen generates the image after the beam strikes it.

Protocol

Method for the visualization of tissue sections (Burghardt. & Droleskey, 2006)

Negative staining

  1. Attach a piece of Parafilm on a clean benchtop with wax-side-down.
  2. With the help of a pipette, place a 15- to 25-μl drop of the sample, two or three droplets of Milli-Q-purified water, and one droplet of the negative staining solution, with a spacing of ∼5 cm, onto the Parafilm.
  3. With the help of self-locking forceps, place a carbon-coated grid on the sample droplet for 5 to 60 sec. Remove the excess liquid between the droplets using the filter paper.
  4. Transfer the grid to the negative stain solution surface for 5 to 30 sec, then remove the grid and dry the excess staining solution by touching the grid to the filter paper wedge.
  5. Air-dry the grid completely to avoid any damage to the sample.
  6. Expose the grids to UV radiation for 10 minutes (if required).
  7. Store the grid at room temperature.

Immunolocalization

  1. Mix 25 μl sample suspension with primary antibody dilution in immunogold dilution buffer in a well of a microtiter plate. Incubate the mixture for 1 hour at 25 to 37◦C in a humidified chamber.
  2. Add 25 μl/well of protein A, protein G, or protein A/G–gold solution and incubate for 30 minutes to 1 hour at 25 to 37◦C in a humidified chamber.
  3. With the help of self-locking forceps, pick up the grids and place them on top of plastic-backed bench protectors with support film facing upward in a row.
  4. Deposit 10 to 15 μl of the mixture from step 2 on the grid with a pipette and let it stand for 5 minutes.
  5. Place the Parafilm with wax-side-down on a clean benchtop.
  6. Place two drops of immunogold antibody dilution buffer and one drop of distilled water with a spacing of ∼5 cm, on the wax surface of the Parafilm. Before drying the sample on the grid, wash the samples by immersing in the two drops of buffer and a drop of distilled water. Remove the excess liquid with the help of filter paper.
  7. Perform negative staining, using 2% phosphotungstic acid, and allow the sample-containing grid to interact with the staining solution for 30 seconds.

Tissue fixation

  1. Slice a fresh piece of tissue <1 mm on a polypropylene cutting board with a razor blade.
  2. Place the tissue slices in a fixation vial containing freshly prepared primary fixative (glutaraldehyde) at a volume 20 times greater than that of the tissue pieces.
  3. Incubate the tissues at room temperature for 60 minutes, then move them to a 4◦C ice bath for 60 minutes.
  4. Decant the fixative from the vials and add phosphate/sucrose rinse buffer at 4◦ Replace the buffer with fresh phosphate/sucrose rinse buffer several times for 30-minutes intervals to wash the tissue.

Post-fixation

  • Fix the tissue pieces in osmium tetroxide for 90 minutes at 4◦ Place the vials on a platform rocker at low speed to facilitate osmium penetration.
  • Decant the osmium solution into a waste container. Rinse the tissue pieces twice in chilled phosphate/ sucrose rinse buffer, then rinse them six times in cold distilled water to remove the phosphate buffer residue.
  • Stain the tissue en bloc in uranyl acetate staining solution for 90 to 120 minutes at 4◦C with periodic agitation.
  • Wash the tissues with distilled water three times to remove uranyl acetate.

Dehydration

  • Dehydrate the samples with ethanol series with the following solutions for 10 minutes each:
    • 50% ethanol, 4◦C
    • 75% ethanol, 4◦C
    • 95% ethanol, room temperature
    • 100% ethanol, room temperature.
  • Rinse the tissue three times for 10 minutes each, in acetone or propylene oxide.

Tissue embedding

  1. Remove acetone or propylene oxide from the fixation vial and replace it with a mixture of 50% (v/v) Luft’s epoxy mixture and 50% acetone. Using an orbital shaker, suspend the tissue pieces or pellet in the resin.
  2. Incubate the samples at room temperature for 30 minutes with rotation on the orbital shaker to facilitate infiltration.
  3. Remove the 50-50 resin mixture and replace it with a mixture of 75% (v/v) Luft’s epoxy mixture and 25% acetone.
  4. Incubate the samples at room temperature for 30 minutes with rotation to facilitate infiltration.
  5. Remove the resin and replace it with 100% Luft’s epoxy mixture. Rotate on an orbital shaker for 1 hour at room temperature, then replace the mixture with freshly prepared 100% Luft’s epoxy mixture.
  6. Remove the resin and transfer the pellets to suitable embedding molds containing freshly prepared 100% Luft’s epoxy mixture. Incubate the molds at 60◦C for overnight.

Sectioning

  1. Cut the tissue into thick sections (0.5-1.0 um), transfer them to a drop of water on a slide and dry it on the slide warmer.
  2. Immerse the slide in Toluidine blue stain for 2-5 minutes.
  3. Observe the tissue sections under the microscope for the precise location to cut ultrathin sections.
  4. Cut ultrathin sections with 60-90 nm thickness and pace the sections onto grids.
  5. Dry the sections overnight before staining.

Staining

  1. Stain the grids containing tissues with uranyl acetate for 15 minutes and lead citrate for 5 minutes.
  2. Observe the slides under the transmission electron microscope.

Protocol for the imaging of bacterial surface structures (Morgellin, 2017)

Culturing

  1. Culture the bacteria to mid-logarithmic phase in Todd- Hewitt broth and incubate at 37 °C with 5 % CO2.
  2. Wash the bacteria and resuspend in Tris-glucose to get a suspension of 2 × 107 cfu/ml.

Grid preparation

  1. Cleave the mica discs and put them on the rotating stage of a carbon coating device with double-sticky tape.
  2. Evaporate a 5 nm carbon film under rotation on the mica surface from a carbon source in a high vacuum (≤10 −6 bar).
  3. Float off the carbon film on the mica disc by emerging the disc in ultra-pure water.
  4. Pick the floating carbon film with the help of electron microscopy copper grids from below and air dry it.

Incubation and conjugation

  1. Incubate 100 μl of the bacterial sample solution with 2–5 μM purified C1q or C3 for 30 minutes.
  2. Wash the sample with Tris-glucose and resuspend the bacterial pellet in the Tris-glucose buffer.
  3. Incubate the bacterial sample with gold-conjugated antibodies against M1 protein (5 nm gold), with anti-C1q (10 nm gold) or C3 (10 nm gold), for 1 hour at room temperature.
  4. Wash with 10 mM Tris–HCl containing 5 mM glucose, and perform negative staining.

Negative staining

  1. Place rows of drops of 1× Tris-buffered saline TBS, 1× bacterial solution, 2× water, 2× uranyl formate, and 1× water on the Parafilm sheet.
  2. Place the grids in a glow discharge device for hydrophilic glow discharge for 30 seconds.
  3. Absorb, wash, and stain the specimens by touching the surface of the different drops for 30–60 seconds each.
  4. Wash the specimen with water, remove the water by blotting the grid on a filter paper, and then air-dry the grids.

Transmission electron microscopy

  1. Observe the negatively stained specimens under the transmission electron microscope at 60 kV accelerating voltage.
  2. Record the images with a side-mounted camera.

Applications

1. Imaging and quantitation of extracellular vesicles (Linares., Tan., Gounou., & Brisson., 2017)

Extracellular vesicles (EVs) are found in blood and other body fluids. EVs are known for their diverse physiopathological roles and biomedical applications.

The characterization and quantification of EVs pose major challenges because of their small size and the lack of methods adapted for their study. Transmission electron microscopy has made significant contributions to the discovery and research of extracellular vesicles.

The TEM combined with receptor-specific gold labeling provided a detailed description of the size, morphology, distribution, and phenotypes of the main extracellular vesicle populations present in platelet free plasma (PFP) as well as different body fluid samples.

2. Characterization of soft materials (Franken., Boekema., & Stuart, 2017)

Transmission electron microscopy (TEM) enables direct structural analysis of nanostructured materials and is a powerful tool for the characterization of soft matter and supramolecular chemistry. It allows the imaging of a large range of objects, from biological systems, e.g., cells or proteins, to materials, extracellular vesicles, and aggregating materials.

The samples were analyzed using the drying, freeze-drying, negative or positive staining, embedding followed by sectioning, quick-freeze deep TEM, and cryo-TEM.

Transmission electron microscopy is the best-suited method for nanomaterials characterization, and the use of this technique could avoid over-interpretations. The use of transmission electron microscopy is leading to further advancement in the structural analysis of biological samples and soft materials.

3. Diagnosis of primary ciliary dyskinesia (PCD) (Shoemark, 2017)

Primary Ciliary Dyskinesia (PCD) is a heterogeneous genetic anomaly characterized by motile cilia dysfunction. The diagnosis of PCD is confirmed by the identification of ciliary ultrastructure defects or by the detection of biallelic pathogenic mutations in the PCD gene.

The assessment of ciliary ultrastructure by transmission electron microscopy (TEM) has been central to its diagnosis and research. The transmission electron microscopy provides improved spatial information and resolution compared to the single micrograph.

Electron microscopy has rendered new insight into the ciliary ultrastructure and ciliary function at a molecular and cellular level.

4. Imaging of liposomes (Baxa, 2018)

Liposome size and size distribution have a major effect on its use as a drug molecule or a diagnostic tool. Transmission electron microscopy is one of the most frequently used methods to characterize the ultrastructure of liposomes.

The liposomal particles are adsorbed on a carbon film grid, surrounded by a heavy metal salt, and air-dried during which the heavy metal salt embeds the particles of interest in the process. This grid is then imaged under the transmission electron microscope where the liposomes appear bright against the dark background of the heavy metal stain.

The transmission electron microscopy provided high-contrast images of the liposomes. The TEM has been widely used for the assessment of liposomes and nano-materials.

Precautions

  • The researcher should wear lab coats and gloves while handling the radioactive material.
  • It is essential to understand the manual of the transmission electron microscope before starting the experiment.
  • The equipment should be monitored for x-ray leakage before and after the experiment.
  • Make sure the samples are appropriately fixed and have retained their structure and optimal conditions.
  • To obtain a TEM analysis, the samples need to be sliced thin enough for electrons to pass through.
  • Clean and dry the samples before placing them in the vacuum chamber.

Strengths and Limitations

  • The transmission electron microscopy allows a detailed analysis of specimens with 500,000X magnification and 0.5 nm resolution.
  • The transmission electron microscope offers an excellent resolution of the crystallographic structures and details of specimens, and could even show the arrangement of atoms within the sample.
  • The TEM could be used for the diagnosis of several pathological conditions as it can unveil the ultrastructural details of the specimen.
  • A Transmission Electron Microscope is a valuable imaging tool for different fields such as life sciences, medical, biological and material research, nanotechnology, forensic analysis, metallurgy, and industry.
  • As the TEM requires the placement of specimens in a vacuum chamber so it cannot be used to visualize the living cells such as protozoa without prior staining.
  • The samples for TEM are limited to the electron transparent materials only that are able to tolerate the vacuum chamber.

References

  1. Baxa, U. (2018). Imaging of Liposomes by Transmission Electron Microscopy. Methods Mol Biol, 1682, 73-88.
  2. R. Burghardt., & Droleskey, R. (2006). Transmission electron microscopy. Curr Protoc Microbiol, Chapter 2:Unit 2B.1.
  3. E. Franken., E. J. Boekema., & Stuart, M. C. (2017). Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation. Adv Sci (Weinh), 4(5).
  4. Morgellin, M. (2017). Negative Staining and Transmission Electron Microscopy of Bacterial Surface Structures. Methods Mol Biol., 1535, 211-217.
  5. Linares., S. Tan., C. Gounou., & Brisson., R. A. (2017). Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy. Methods Mol Biol, 1545, 43-54.
  6. Shoemark, A. (2017). Applications of emerging transmission electron microscopy technology in PCD research and diagnosis. Ultrastruct Pathol, 41(6), 408-414.
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