Electron Microscopy

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Introduction

An electron microscope is an imaging instrument that uses a beam of energetic electrons to observe objects on a very fine scale. Electron Microscopes were developed to overcome the limitations of light microscopes which can only visualize the specimens with 500x or 1000x magnification and 0.2 micrometers resolution. In the early 1930’s the scientific desire to visualize the fine details of the intracellular components and organelles rose. This required 10,000x plus magnification which led to the development of the electron microscope. Max Knoll and Ernst Ruska developed it in Germany in 1931. In the electron microscope, a focused beam of electrons is used to “see-through” the specimen. This examination can yield information about the surface features of an object, morphology, composition and crystallography.

The basic steps of the electron microscopy involve; the electron gun generates a stream of highly energized electrons. This stream is bombarded onto the specimen (with a positive electrical potential) while is limited and focused with the help of metal apertures and magnetic lenses into a thin, focused, monochromatic beam. This beam irradiates the sample and interactions inside the irradiated sample occur, affecting the electron beam. These interactions and effects are captured and transformed into an image.

Principle

In an electron microscope, an ‘electron beam’ is used to produce the image and the ‘electromagnetic field’ is used for the magnification. Electrons are subatomic particles orbiting around the nucleus. Electrons fly off form the atom after the metal is excited by heat. In the electron microscope, tungsten is heated by a high voltage current to form a continuous electron stream. Magnetic coils are used in the electron microscope to focus the beam on the specimen and illuminate it.

The wavelength of light is inversely proportional to the resolving power. The wavelength of green light is 1,10,000 times greater than that of an electron beam; therefore; despite its smaller numerical aperture the electron microscope can resolve objects as small as 0.001µ. This makes the resolving power of the electron microscope 200 times greater than that of the light microscope.

Apparatus

The electron microscope consists of an electron gun, electromagnetic lenses, and an image viewing and recording system. The electron gun is a heated tungsten filament which generates an electron beam. The condenser lens focuses this beam on the sample. A second condenser lens is used to form a thin tight electron beam. An accelerating voltage is applied to move electrons down the column. The highly energized electrons pass into the condenser lenses, which then focus them on the specimen. The electrons pass through the specimen and are scattered depending upon the thickness of the different regions of the specimen. The scattered electrons come out of the specimen and pass down to the magnetic coils called the objective lens, which forms the intermediate magnified image. Finally, the other set of magnetic lenses called projector or ocular lenses produce the final magnified image.

Protocol

Cell fixation

1. Fix the cells in a mixture of 25% formaldehyde, 2.5 % glutaraldehyde and 0.03% picric acid mixed in 0.1 M Sodium cacodylate buffer. Add the fixative (2x concentration) in a 1:1 ratio to the cell media. Leave it for 1 hour at room temperature.
2. Wash the cells thrice in cacodylate buffer.
3. Add 1% Osmium tetroxide/1.5% Potassium ferrocyanide (in H₂O) and leave for 1 hour at room temperature.
4. Wash the cells with water three-four times.
5. Add 1% Uranyl Acetate in water and leave for 30 minutes to 1 hour.
6. Wash the cells in water three times.

Dehydration

7. Dehydrate the cells in 70% Ethyl alcohol for 15 minutes, then in 90% Ethyl alcohol for 15 minutes, and after that in 100% Ethyl alcohol 2 times for 15 minutes.
8. Add Propylene oxide and leave for 1 hour.

Infiltration

9. Mix the cells in 1:1 Epon and propylene oxide at 4^o
10. Move the samples to embedding mold filled with freshly mixed Epon.
11. Allow the sample to sink, and move to oven for polymerization.
12. Leave the cells to polymerize for 24-48 hours at 60°C.

Ultra-sectioning

13. Cut the ultra-thin tissue slices with the help of a diamond knife or glass knives.

Staining

14. Stain the cells with heavy metal as per the experimental needs.
15. Mount the slides with the cells on the microscope and observe.

Protocol for the detection of viruses (Goldsmith. & Miller., 2009)

1. Inactivate the viruses by treating the viruses with 2–4% paraformaldehyde dissolved in phosphate buffer saline.
2. Administer the droplets (30 μl) of the different solutions onto the clean surface and incubate the filmed grids on them.

Note: Perform all the steps on a desk covered with a strip of Parafilm.

3. Incubate the filmed surface of the grid in Alcian blue solution (1% in 1% acetic acid) for 10 minutes.
4. Wash the filmed surface by sprinkling four droplets of double-distilled water.
5. Remove the surplus water with the help of a filter paper.
6. Incubate the filmed surface in the sample suspension for 10 minutes.
7. Wash the filmed surface by successively touching with the four droplets of double-distilled water.
8. Remove the residual water on the grid surface with the help of a filter paper.
9. Touch a drop of the negative stain (1% uranyl acetate in double-distilled water) for a few seconds.
10. Carefully remove the staining solution with a filter paper to form a thin homogeneous film of stain.
11. After a brief drying period (a few minutes are sufficient), analyze the samples under the electron microscope.

Applications

Visualizing autophagy in mammalian cells (Anttila., Vihinen., Jokitalo., & Eskelinen., 2009)

The electron microscopy is a powerful imaging tool to delineate the cellular events involved in autophagy. The cells were prepared for electron microscopy and embedded with resins. Under the electron microscope, autophagic compartments are observed as membrane-bound vesicles loaded with cytoplasmic material or organelles. Ribosomes were frequently seen in the autophagic vesicles. High stain retaining in the limiting membrane suggested that the membrane has a high content of unsaturated lipids. The study validated the value of electron microscopy in studying the cellular events for biomedical research.

Diagnostic electron microscopy for retina (Gupta., Kaur., Nag., & Chhablani., 2018)

The electron microscopy has been used in various fields as a diagnostic tool in vitro, which provides efficient resolution for better interpretation and imaging. A number of studies have been done employing the electron microscopy to visualize the fine cellular and subcellular structure such as photoreceptors (rod and cone cells), retinal pigment epithelium (RPE), outer plexiform layer, inner plexiform layer, bipolar cells, ganglion cells, and nerve fiber layers. Furthermore, the electron microscopy has successfully been used to reveal the pathological conditions such as light-induced photoreceptor degeneration, age-related muscular degeneration, synaptic plasticity, and other retinal pathologies. The electron microscopy is a very promising technique to decipher the molecular mechanism of the pathological state, the conformational changes of the proteins, and the visualization of the cellular ultrastructure.

Imaging the virus-cell interactions (Bykov., Cortese., Briggs., & Bartenschlager., 2016)

The electron microscopy combined with the light microscopy has wide applications in visualizing and characterizing the morphological and conformational alterations of the viral replication cycle. The cells were prepared and infected with the viruses. The electron microscopy revealed the remodeling of the viral core and the rearrangement of the surface glycoproteins of the virus to shape the entry claw upon host receptor binding. The assembly, maturation, and the release processes complete the viral replication cycle. It was found that the assembly of non-enveloped viruses occurs in the cytoplasm or the nucleus of the cell and they lyse the cell for release. In contrast, the enveloped viruses attain a lipid bilayer that is derived from cellular membranes. The electron microscopy provides detailed mechanistic insights into the processes of viral replication cycle that will play an important role in providing spatiotemporal parameters of biological processes and help to unveil the molecular composition of involved molecular complexes.

Diagnosing the neuromuscular diseases (Goebel. & Stenzel., 2013)

The electron microscopy is a useful imaging tool for the diagnosis of neuromuscular conditions. The electron microscopy provides with a specific diagnosis by unveiling the ultrastructural features such as inclusions within muscle fibers, cylindrical spirals, and reducing bodies of the neuromuscular pathological states. The technique could also aid in the diagnosis of muscular dystrophies, neurogenic atrophy, myotonic diseases, and congenital, metabolic, and inflammatory myopathies.

Precautions

• Handle the chemicals in the fume hood.
• Wear disposable gloves while preparing the mixtures.
• Cover the working areas with a paper towel and immediately wipe spills with alcohol.
• Most vacuum evaporators do not have a safety switch to turn off power before opening the bell jar.
• Never observe the metal evaporation without goggles. The intense brightness can burn the retina.
• Do not observe the critical dryers without understanding the associated danger.
• Carefully clean the microscope before and after the experiment.

Strengths and limitations

• The electron microscopy offers a higher resolution than other imaging techniques and plays an important role in biomedical science.
• Electron microscopy has a diverse range of applications in industry, technology, and scientific research such as semiconductor inspection, computer chips manufacturing, quality control, and product testing.
• An electron microscope is a promising tool for the observation of minute details within cells, such as tissue arrangements and the positions of organelles.
• Electron microscopes are used for a deeper understanding of the structure-property-function relationships in a broad range of samples and processes such as solar cell technologies, catalyst activity, and chemical sensitivity.
• The main disadvantages of the electron microscopy include higher cost, large size, heavy maintenance, researcher training, and image artifacts.

References

1. S. Goldsmith., & Miller., S. E. (2009). Modern Uses of Electron Microscopy for Detection of Viruses. Clin Microbiol Rev, 22(4), 552-563.
2. H. Goebel., & Stenzel., W. (2013). Practical application of electron microscopy to neuromuscular diseases. Ultrastruct Pathol, 37(1), 15-8.
3. K. Gupta., I. Kaur., T. C. Nag., & Chhablani., J. (2018). Diagnostic Electron Microscopy of Retina. Semin Ophthalmol, 33(5), 700-710.
4. P. Anttila., H. Vihinen., E. Jokitalo., & Eskelinen., E. L. (2009). Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol, 452, 143-64.
5. S. Bykov., M. Cortese., Briggs., J. A., & R. B. (2016). Correlative light and electron microscopy methods for the study of virus-cell interactions. FEBS Lett, 590(13), 1877-95.