An electron microscope is a type of microscope that uses an electron beam for illumination and creates an enlarged image of the specimen. (In contrast, light microscope uses visible light for purpose of illumination).
An electron microscope has great resolving power and can create much higher magnification as compared to a light microscope. Hence it enables finer details of smaller objects to be seen clearly. Its greater resolution and magnification is due to the fact that the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons.
Some electron microscopes are capable of magnifying specimens up to 2 million times, while the light microscopes can show a maximum magnification of 2000 times.
It is used in the medical field for research purposes, to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes. It provides key information about the structural basis of cell function and cell disease. In conjunction with a variety of ancillary techniques, it can be used to diagnose various tumors and non-tumor conditions.
Examination of ultrastructural features of cellular and extracellular structures is a powerful diagnostic tool. The introduction of the transmission electron microscope in the early decades of the twentieth century dramatically expanded the investigative and diagnostic capabilities to study the submicroscopic details of diseased tissue.
Types of Electron Microscopy
Transmission electron microscopy (TEM)
It is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.
Scanning electron microscopy (SEM)
A scanning electron microscope produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected. It gives information about the sample’s surface topography and composition. The resolution obtained by SEM is poorer as compared to TEM. However, it can create images of large samples and has a greater depth of field. So images from SEM are good representations of the real shape of the specimen.
To view a sample under the electron microscope, it must be processed in a proper way. The various steps of processing include
The specimen is frozen rapidly to liquid nitrogen temperatures or below. This preserves the specimen producing minimal artifacts.
It is done to prevent further deterioration of the material or tissue. Glutaraldehyde is often used to fix protein molecules and osmium tetroxide to preserve lipids.
Water is removed from the samples. Organic solvents such as ethanol or acetone are used for SEM specimens while infiltration with resin is carried out for TEM specimens.
The tissue is then infiltrated with resin and a hardened block is prepared for subsequent sectioning.
Very thin slices of the specimen are then cut (90 nm thick) with an ultramicrotome using a glass or diamond knife.
It is done using heavy metals such as lead and uranium. Staining helps to scatter imaging electrons and produces contrast between different structures.
It is used for a variety of purposes including high-resolution imaging, 2D & 3D micro-characterization, experiments on dynamic materials, particle detection and characterization, sample preparation, metallurgy, nanotechnology, petrochemical industry, semiconductor inspection, computer chip manufacturing, etc
Qualification and sample preparation of materials, nanoprototyping, nanometrology, device testing, and characterization, etc.
Biology and life sciences
- Protein localization
- Electron tomography
- Cellular tomography
- Cryo-electron microscopy
- Biological production and viral load monitoring
- Particle analysis
- Pharmaceutical quality control
- 3D tissue imaging
Diagnostic uses in medicine
Ultrastructural diagnosis of non tumor biopsies
- Diseases of kidney
- Metabolic storage diseases
- Respiratory tract diseases
- Skeletal muscle diseases
- Infectious agents
- Skin disorders
- Peripheral nerve biopsies
Ultrastructural diagnosis of tumors
- Epithelial tumors
- Hematopoietic tumors
- Lymphoid tumors
- Soft tissue tumors
- Small round cell blue tumors
- Central nervous system tumors
Electron microscopy is a useful investigation for diagnosing tumors in conjunction with light microscopy and immunohistochemistry. Though not routinely performed for diagnosing tumors in all cases, it can provide useful information in troublesome and difficult to diagnose cases. Its main uses in tumor diagnosis include:
- To confirm light microscopic diagnosis of tumor
- Differentiating primary and metastatic tumors
- Finding organ of origin in case of a metastatic tumor with unknown primary
- Evaluating undifferentiated malignant neoplasms
- Subtyping sarcomas, leukemias and lymphomas
An electron microscope is an expensive instrument. Both the initial purchasing cost and cost of maintenance are very high.
Regular power consumption
It is a dynamic instrument which means that it requires an extremely stable high voltage. Each electromagnetic coil or lens needs a constant and steady current.
The various parts of electron microscope require a high degree of maintenance. For eg, the cooling system needs constant circulation pumping through the unit while the vacuum setup requires consistent pressure and continuous pumping.
It is a very sensitive instrument. It should be placed in a special area and must be protected from vibrations and external magnetic fields.
Preparation of the sample is a time consuming and labor intensive process. Also the samples need to be viewed in a vacuum otherwise naturally occurring molecules in the air can scatter and distort the electrons.
Both the sample preparation and interpretation require highly skilled and trained professionals.
Artifacts and errors
Sample manipulation during processing can result in artifacts or inadvertent changes in the structure of the specimen. These can produce erroneous readings and interpretations.