How are samples prepared for a transmission electron microscope?

What is transmission electron microscope?

A transmission electron microscope (TEM) is a powerful scientific instrument that is used to study the structure and properties of materials at the atomic and molecular level. It works by using a beam of electrons to create an image of a sample, which is then analyzed to study the sample’s structure and properties.

TEMs are used in a wide variety of fields, including materials science, biology, and nanotechnology, to study the structure and properties of materials at the atomic and molecular level. They are particularly useful for studying materials that are too small, too transparent, or too opaque to be studied with an optical microscope.


TEMs are typically larger and more complex than other types of microscopes and require a dedicated laboratory space. They consist of several components, including an electron gun, which generates the beam of electrons, a vacuum system, which maintains a high vacuum inside the microscope column, and an electron lens, which focuses the beam of electrons onto the sample.

Overall, a transmission electron microscope is a powerful scientific instrument that is used to study the structure and properties of materials at the atomic and molecular level. It is an essential tool for understanding the fundamental properties of matter and has played a key role in many scientific and technological advances.


Why We need to prepare specimen sample for TEM?

There are several reasons why it is important to prepare specimen samples for transmission electron microscopy (TEM):

  1. Sample size and thickness: Samples for TEM should be thin enough to allow the electrons to pass through them and should be mounted on a transparent support, such as a film or a grid. The sample should be no thicker than about 100 nm, and samples that are too thick may need to be thinned using mechanical or chemical methods.
  2. Sample preparation techniques: There are several techniques that can be used to prepare samples for TEM, including mechanical grinding and polishing, chemical etching, and focused ion beam milling. The specific technique that is used depends on the type of sample and the specific requirements of the experiment.
  3. Specimen holders: Samples for TEM should be mounted on a specimen holder, such as a film or a grid, using a thin layer of glue or other adhesive. The specimen holder should be transparent and should not interfere with the transmission of the electrons through the sample.
  4. Sample preparation in a vacuum: Samples for TEM should be prepared in a vacuum to prevent contamination by air molecules. The sample should be placed in a vacuum chamber and should be handled using gloves or other protective measures to prevent contamination.
  5. High-quality images: Proper sample preparation is essential for producing high-quality images with a TEM. Samples that are not properly prepared may produce images that are distorted or contain artifacts, which can affect the accuracy of the results.
  6. Preservation of sample integrity: Proper sample preparation is also important for preserving the integrity of the sample. Samples that are not properly prepared may be damaged or contaminated, which can affect the accuracy of the results.

Overall, preparing samples for TEM requires careful attention to detail and the use of specialized techniques and equipment. Proper sample preparation is essential for producing high-quality images and for preserving the integrity of the sample.


Sample preparation in TEM

In this method, the particle under study is subjected to electron beams using a transmission electron microscope, and the resulting micrographs or photographs are computationally evaluated.

Sample preparation is a vital stage in transmission electron microscopy (TEM), and the sample preparation procedure varies depending on the nature of the material and the information sought from it.


The preparation of specimens for TEM comprises multiple steps:

1. Fixation

Fixation of the specimen stabilises the cell, preventing future alteration or harm to the cell. This method preserves the sample to provide a snapshot in time of the living cell. Fixation can be achieved by the following two methods:

  • Chemical fixation: Chemical fixing is a technique used to stabilise biological specimens. To cross-link protein molecules with adjacent molecules, chemical chemicals are utilised. Glutaraldehyde is the most often employed chemical in this procedure.
  • Cryofixation: This technique requires the fast freezing of the sample in liquid nitrogen or liquid helium. Thus, the sample’s water content is changed into a kind of vitreous ice.

2. Rinsing

The procedure of tissue fixation may raise the acidity of the specimen. To prevent this state and preserve the pH, it must be carefully rinsed with a buffer, such as sodium cacodylate, to retain its balance.

3. Secondary fixation

Osmium tetroxide is used to boost the contrast of the minute features inside the specimen and to provide more stability (OsO4). OsO4 turns proteins into gels and raises the contrast between neighbouring cytoplasm by binding sections of phospholipid heads without altering the structure’s characteristics.


4. Dehydration

Freeze-drying, or dehydration, is the replacement of the specimen’s water content with an organic solvent. In this process, ethanol and acetone are the most commonly utilised solvents. Important because the epoxy resin used in subsequent processes does not mix with water.

5. Infiltration

In infiltration, epoxy resin is injected into the cell to fill the space and make the sample sufficiently rigid to withstand the pressure of sectioning or cutting. This method is also known as embedding. The resin is then placed in a 60-degree oven overnight for setting. This method is known as polymerization.

6. Polishing

After embedding, certain materials are treated to polishing. Polishing a specimen minimises scratches and other issues that can diminish the image’s quality. The specimen is polished using ultrafine abrasives to achieve a mirror-like surface.

7. Cutting

For examination under an electron microscope, the sample must be semi-transparent to allow electron beams to flow through it. To acquire this semi-transparent quality, the sample is sectioned using a glass or diamond knife attached to an ultramicrotome instrument. The apparatus includes a trough filled with distilled water.

The slices are gathered in this trough before being transferred to a copper grid for examination under a microscope. For optimal resolution, the size of each section should be between 30 and 60 nanometers.

8. Staining

Staining is typically performed twice on biological specimens, before dehydration and after sectioning. Heavy metals such as uranium, lead, or tungsten are utilised in this procedure to heighten the contrast between distinct structures in the specimen and scatter the electron beams.

Before hydration, the sample is stained in block, whereas after sectioning, it is temporarily exposed to an aqueous solution of the aforementioned metals.

A specimen that has been cryofixed may not undergo all of these treatments. It can be directly exposed to cutting and then shadowed with platinum, gold, or carbon vapours prior to TEM imaging.

In addition to the above general steps for preparing a sample for TEM, other alternative techniques are available, such as:

  1. Ion-mining: In this method, the sample is thinned by firing charged argon ions at its surface until it becomes sufficiently transparent. The process of focussed ion mining utilises gallium ions for tinning.
  2. Cross-sectional method: This technique is mostly used to examine interfaces.
  3. Replica technique: Used only when the bulk specimen used to create thin sections cannot be destroyed.
  4. Electrolyte polishing: Electrolyte polishing is a technique used to create thin metal or alloy samples. Various techniques, such as coring, rolling, grinding, and peeling, are incorporated in this approach.

Conventional Method For preparation of ultrastructure

The following stages comprise a standard technique for preparing samples for ultrastructure imaging.

1. Primary attachment to aldehydes (proteins)

During this phase, formaldehyde and/or glutaraldehyde molecules crosslink proteins and, to a lesser extent, other cell components. Fixation of small mammals is possible by perfusion, in which the fixative is administered via the circulatory system. Other samples must be fixed by immersion, and the specimen must be dissected in at least one direction to a maximum thickness of 1 mm.

2. Secondary fixation with osmium tetroxide (lipids)

This process guarantees that lipids, such as membrane-forming phospholipids, are retained and not removed during dehydration. During the fixation process, a black, insoluble precipitate is generated on the membranes, generating contrast.

3. Tertiary fixation and contrasting with uranyl acetate

Uranyl acetate is a heavy metal salt that provides extra contrast by binding to proteins, lipids, and nucleic acids. Some authors argue that it also possesses fixative characteristics. Before dehydration, samples can be incubated in a solution of uranyl acetate, or the stain can be applied to sectioned specimens prior to lead staining.

4. Dehydration series with solvent (ethanol or acetone)

By incubating a specimen in a series of ethanol or acetone solutions, it is dehydrated. The concentration of the solvent is increased gradually so that water can be removed without generating artefacts, primarily shrinking.

5. Resin infiltration and embedding

Following dehydration, a progressively increasing concentration of liquid resin is substituted for the solvent (typically epoxy resin for ultrastructure studies). The specimen is placed in a mould containing liquid resin, which is then hardened using heat or ultraviolet radiation. A sample can then be stored indefinitely.

6. Sectioning and mounting sections on specimen grids

A specimen immersed in a solidified resin can be sectioned to a thickness of less than 100 micrometres. This allows the electron beam to travel through the specimen from the electron gun to the detector. The sections are mounted on specimen grids that fit into the sample holder of the microscope.

7. Contrasting (poststaining)

Since biological specimens are formed of atoms with low atomic numbers, they are naturally not highly electron-opaque, and the electron beam can easily pass through them. The sections can be post-stained with lead citrate to enhance contrast. Similar to osmium tetroxide and uranyl acetate, this heavy metal salt binds to cell components and scatters incident beam electrons. The parts of the specimen section that scatter electrons more are represented by pixels with a deeper hue, which stand out against the lighter backdrop.



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