Transmission Electron Microscopy, or TEM, is an imaging technique that uses a beam of electrons instead of light to visualize objects at an extremely high level of detail. This method allows scientists to see structures far smaller than what a conventional light microscope can resolve. By passing electrons through a sample, TEM reveals the internal features of specimens, enabling detailed analysis of objects at the nanoscale. Its capability to achieve magnifications of over one million times makes it a foundational tool in many scientific fields.
The Inner Workings of a Transmission Electron Microscope
At the top of a transmission electron microscope’s column sits an electron gun, which is the source of the electron beam. This component contains a filament, often made of tungsten, that releases a stream of electrons when heated. These electrons are then accelerated by a high voltage, ranging from 80 to 300 kilovolts (kV), giving them the energy needed to travel through the microscope’s column. The entire column is kept under a high vacuum to prevent the electrons from colliding with air molecules, which would scatter the beam and disrupt the image formation process.
Once the electron beam is generated, it is guided and focused by a series of electromagnetic lenses. Unlike the glass lenses in a light microscope, these are magnetic coils that create precise fields to control the electron path. Condenser lenses are used to concentrate the beam onto the specimen. After the electrons pass through the sample, another set of lenses, including the objective, intermediate, and projector lenses, work together to magnify the resulting image.
The image is formed based on how the electrons interact with the specimen. As the beam passes through the ultra-thin sample, some electrons are scattered by atoms within the material, while others pass through unimpeded. Denser parts of the sample, or areas containing heavier elements, scatter more electrons and therefore appear darker in the final image. The transmitted electrons are projected onto a fluorescent screen or captured by a digital camera, creating a high-resolution, two-dimensional black-and-white image.
Preparing a Sample for Viewing
A specimen must undergo extensive preparation before it can be viewed in a transmission electron microscope. For biological samples, the first step is fixation, which preserves the tissue and prevents decomposition. This is achieved using chemical fixatives like glutaraldehyde, which cross-links proteins and stabilizes the cellular structure. Following fixation, the sample is rinsed and then dehydrated by immersing it in a graded series of ethanol or acetone to remove all water.
After dehydration, the sample is infiltrated with a liquid epoxy or resin that permeates the cells and tissues. This resin is then hardened through a process called polymerization, embedding the biological material in a solid block that provides support. This step is necessary for the specimen to withstand the vacuum inside the microscope and the electron beam. The solid block makes it possible to cut extremely thin sections from the sample.
Using a specialized instrument called an ultramicrotome, fitted with a diamond or glass knife, the embedded sample is sliced into sections that are thin, often between 30 and 100 nanometers. This thinness is required to allow the electron beam to pass through the specimen. To generate contrast in the final image, these sections are stained with solutions containing heavy metals, such as uranyl acetate and lead citrate. These heavy atoms preferentially bind to different cellular structures, increasing their electron density and making them appear darker in the TEM image.
Visualizing the Nanoscale World
The high resolution of transmission electron microscopy makes it a valuable tool in biology and medicine. It allows researchers to visualize the internal ultrastructure of cells, revealing the morphology of organelles such as mitochondria, ribosomes, and the endoplasmic reticulum. This capability is useful in virology, where TEM can be used to directly observe and identify individual virus particles, which are too small to be seen with a light microscope. The images help in understanding viral structure and replication, aiding in the diagnosis and study of diseases.
In the fields of materials science and nanotechnology, TEM provides insights into the structure of matter at the atomic level. Scientists use it to examine the arrangement of atoms in a crystal lattice, identify defects or imperfections in materials, and characterize the size and shape of nanoparticles. This information helps in developing new materials with specific properties, analyzing failures in semiconductor devices, and advancing the manufacturing of micro-sized objects.
Comparison with Scanning Electron Microscopy
A common point of confusion is the difference between transmission electron microscopy and scanning electron microscopy (SEM). The primary distinction lies in how the electron beam interacts with the sample. In TEM, the electron beam passes directly through an ultra-thin specimen to form an image. Conversely, SEM scans a focused beam of electrons across the surface of a sample, and detectors collect the electrons that are scattered or reflected off that surface.
This operational difference results in distinct types of images. TEM produces a two-dimensional, high-resolution image of the internal structure of a sample, similar to a cross-section. SEM, on the other hand, generates a three-dimensional-like image that reveals the surface topography and texture of the specimen. This makes SEM suitable for observing the surface features of an object, while TEM is used to study its internal composition.
The sample requirements for each technique also differ significantly. TEM necessitates that samples be extremely thin, under 100 nanometers, to allow electrons to be transmitted through them. Preparing these thin sections can be a complex and time-consuming process. In contrast, SEM can be used to analyze thicker, bulk samples without the need for extensive sectioning, although samples often need to be coated with a conductive material like gold to prevent electrical charging. The choice between TEM and SEM depends on the information sought, whether it is the internal ultrastructure or the external surface features of a specimen.