A transmission electron microscope (TEM) is a scientific instrument that creates highly magnified images of a sample’s internal structure using a beam of electrons. The term “transmission” refers to the process where these electrons pass through the specimen, allowing for the visualization of features from micrometers down to nanometers.
Core Principle of Operation
The process begins with the generation of electrons inside an electron gun, which can be one of two main types: thermionic or field emission. In a thermionic gun, a filament is heated until it releases electrons. These electrons are then accelerated by a high voltage, typically ranging from 80 to 300 kilovolts (kV), which gives them very high energy and a short wavelength, enabling high-resolution imaging.
This beam of high-energy electrons travels down a column that is kept at a high vacuum. The vacuum is necessary because electrons are easily deflected by air molecules, which would scatter the beam and prevent a clear image from being formed.
As the accelerated electrons travel toward the sample, they pass through a series of electromagnetic lenses. These lenses, including a condenser lens system, use magnetic fields to shape and focus the electron beam onto the specimen. The objective lens performs the initial magnification and is a primary determinant of the final image’s resolution.
When the focused beam strikes the ultrathin specimen, some electrons pass directly through it, while others are scattered by the atoms within the sample. Denser parts of the sample, or areas treated with heavy metals, scatter more electrons. This differential scattering is the basis of image contrast; areas where many electrons are scattered appear dark in the final image, while areas where electrons pass through unimpeded appear bright.
Finally, the transmitted electrons are projected onto a detector. This can be a fluorescent screen, photographic film, or a digital camera, such as a charge-coupled device (CCD). The pattern of bright and dark areas forms a two-dimensional, black-and-white image known as a micrograph, which reveals the internal ultrastructure of the specimen.
Sample Preparation for Viewing
Because electrons have limited ability to penetrate materials, specimens must be made exceptionally thin, between 50 and 100 nanometers. For biological samples, this process begins with fixation, a step that preserves the cellular structure in a life-like state using chemicals such as glutaraldehyde and osmium tetroxide.
Once fixed, the water must be removed from the specimen through a process of dehydration, which is usually accomplished by immersing it in a series of increasingly concentrated ethanol solutions. To make the soft, dehydrated tissue solid enough to be sliced thinly, it is infiltrated with a hard epoxy resin. This embedded sample is then ready for sectioning.
The hardened block containing the sample is cut using an ultramicrotome, which is equipped with an extremely sharp diamond knife. This tool can slice the sample into sections that are nanometers thick. These ultrathin sections are then carefully mounted onto a small, circular copper grid that will hold them inside the microscope.
Before viewing, the sections on the grid are stained to enhance contrast. TEM stains consist of heavy metal salts, such as uranyl acetate and lead citrate. These electron-dense metals accumulate in specific cellular structures, making details visible. For materials science, different methods like ion milling are used to thin samples.
Applications and Discoveries
In cell biology, the microscope revealed the intricate internal world of the cell, providing the first clear views of organelles. Researchers could see the detailed structures of mitochondria, the endoplasmic reticulum, and the Golgi apparatus, changing our understanding of cellular function.
Virology also owes many of its foundational discoveries to the TEM. Before its invention, viruses were mysterious entities, too small to be seen with light microscopes. The TEM made it possible to directly visualize and photograph virus particles, such as poliovirus, which is only 30 nanometers in diameter. This ability was instrumental in classifying viruses and understanding how they replicate.
In the field of medicine, particularly pathology, the TEM remains a diagnostic tool for certain conditions. Some kidney diseases, for example, are characterized by changes to the ultrastructure of the glomeruli, the filtering units of the kidney. These subtle changes are only visible with the high magnification of a TEM, allowing for precise diagnosis.
Materials science has also been transformed by transmission electron microscopy. Scientists can examine the atomic lattice of crystalline materials, identifying defects, dislocations, and impurities that affect the material’s properties. This analysis is applied in nanotechnology, semiconductor research, and the development of new alloys and composites.
Comparison with Other Microscopes
When compared to a standard light microscope, the most significant differences are the illumination source and achievable resolution. Light microscopes use a beam of light and glass lenses, while a TEM uses a beam of electrons and electromagnetic lenses. This allows the TEM to achieve magnifications far beyond the reach of light microscopy, revealing details at the atomic scale.
A more direct comparison is with the scanning electron microscope (SEM), as both use electrons. The fundamental difference is how they interact with the sample. A TEM forms an image from electrons transmitted through an extremely thin specimen, providing a two-dimensional view of the sample’s internal structure. The SEM, in contrast, scans a focused beam of electrons across the surface of a sample to create a detailed, three-dimensional-like view of its topography. The choice between TEM and SEM depends on whether the research requires viewing internal ultrastructure or examining surface features.