Electron microscopes are a type of microscope that use a beam of accelerated electrons to illuminate a specimen and create a magnified image. They are capable of achieving much higher resolution than light microscopes and are integral in the field of cell biology for the detailed examination of organisms, cells, and cellular components.

Principle of Operation

Electron microscopes function on the same basic principles as light microscopes but differ in that they use a beam of high-energy electrons instead of light to create an image. Electrons have much shorter wavelengths than light, which results in much higher resolution.

The electron beam follows a similar path to that of light in a light microscope but uses electromagnetic lenses instead of glass lenses to focus the beam onto the sample. These lenses are arranged in an "electron optical column." The magnified image can be viewed directly on a screen, printed on photographic paper, or captured digitally.

Transmission Electron Microscope (TEM)

Transmission Electron Microscopes (TEM) were the first type of electron microscope to be developed and are best suited for looking at the internal structure of a sample. They work by firing a high-voltage electron beam through an ultra-thin slice of the specimen. The beam passes through the specimen, with parts of the beam being absorbed and diffracted, and lands on a phosphorescent screen to create an image.

Sample Preparation for TEM

The preparation of samples for TEM analysis can be complex and time-consuming. It generally involves the following steps:

• Fixation: The sample is chemically fixed using a substance like glutaraldehyde or osmium tetroxide to preserve its structure.
• Dehydration: The sample is then dehydrated, usually through a graded series of alcohol, followed by substitution with a medium that is compatible with the epoxy resins used in the next step.
• Embedding: The sample is then embedded in resin, which is then polymerised to harden it.
• Sectioning: The hardened block is then sliced into extremely thin sections, often using a diamond knife in a device called an ultramicrotome. These sections are usually between 50 and 100 nm thick.
• Staining: The sections are then stained with heavy metals like uranium or lead, which scatter the electron beam to different extents, providing contrast in the image.

Interpretation of TEM Images

Images produced by TEMs are 2D projections of the sample. The denser regions that absorb more electrons appear darker, while the less dense areas appear lighter. This contrast allows for different structures within the sample to be distinguished. TEM images can show fine details, including some sub-cellular structures, down to the molecular level.

Scanning Electron Microscope (SEM)

Unlike TEMs, which provide images of the internal structure of a sample, Scanning Electron Microscopes (SEMs) are used to study the surface characteristics and composition of specimens. They work by scanning a focused beam of electrons across the surface of a specimen. The electrons in the beam interact with the atoms in the sample, producing various signals such as secondary electrons and backscattered electrons that can be detected and used to generate an image.

Sample Preparation for SEM

Sample preparation for SEM analysis is generally simpler than for TEM. It usually involves:

• Fixation: Similar to TEM, the specimen is first fixed chemically to preserve its structure.
• Dehydration: The sample is then dehydrated. This can be done using a series of alcohol solutions or by critical point drying to avoid distortion.
• Mounting: The dehydrated specimen is then mounted onto a sample holder, or "stub," using a conductive adhesive.
• Coating: The sample is then coated with a thin layer of conductive material, usually a metal like gold or platinum, using a process called sputter coating. This prevents the sample from charging under the electron beam and improves the generation of signals for imaging.

Interpretation of SEM Images

SEM images are 3D in appearance and show the surface topography of the sample. The brightness of the image corresponds to the number and energy of the secondary electrons emitted from the surface. High spots on the surface emit more secondary electrons and appear brighter, while low areas emit fewer and appear darker. The resulting image can provide a wealth of information about the surface texture, size and shape of surface features, and the distribution of different elements on the surface.

Differences Between TEM and SEM

While TEM and SEM are both types of electron microscopes, they differ significantly in their operation and the type of information they provide:

• TEM provides detailed images of the internal structures within a specimen, while SEM gives detailed images of the surface topography and composition.
• The sample preparation for TEM is more complex and time-consuming than for SEM, as it requires the creation of ultra-thin sections.
• TEM has a higher resolution than SEM, making it more suitable for looking at structures at the molecular level.
• SEM can accept a wider range of sample sizes and types, as it does not require the sample to be ultra-thin.
• The images produced by SEM are easier to interpret, as they are similar in nature to a 3D view seen with the naked eye, while TEM images are 2D projections and require more knowledge and experience to interpret correctly.