Transmission Electron Microscopy: Its Working and Applications

Introduction

In 1900, scientists observed that light microscopy had its own limitations because useful magnification could not be achieved beyond 1000X, with a limited resolution of 0.2 µm or 200 nm. Then, in 1933, Ernst Ruska from Germany noted that the limitation in magnification was due to the limitation of wavelength of visible or UV light, which could not go below 200nm. The wavelength needed to be smaller than the object and its microstructures to resolve them as separate and distinct entities for attaining high useful magnification.

To overcome this hurdle, Ruska turned to "electrons," charged particles that could be accelerated using various voltages (ranging from 60 to 300 KV). This approach allowed him to achieve a wavelength of electron as low as 0.002 nm. Building on these insights, Ernst Ruska and Max Knoll collaborated to develop the first Transmission Electron Microscope (TEM).

What are the applications of TEM?

TEM has numerous applications, using thin sections of the specimen, it helps to detect and observe the interior structures of the cell, cytoskeletal filaments, cell membranes (lipid bilayer), localized transmembrane proteins, cell organelles, mitosis/meiosis at the ultrastructural level for studying cell division, internal structures of bacterial cells, viruses, and their spike receptors (protein) for making vaccines or antiviral drugs.

It is also used to study the mechanisms of disease, characterize nanoparticles used in drug delivery systems, conduct cancer research, and analyse chemical structures of molecules up to atomic levels.

Difference
TEM	SEM
Provide details of internal structures of the specimen	Describes topography or surface morphology of the specimen
Thin sections (<100nm) of the specimen are required to observe internal structures	Section cutting is NOT required. Thick sections can be observed.
Electrons transmit through the specimen	Electrons hit the surface of the specimen and scatter or produce secondary electrons
Due to section cutting sample preparation is complex and tedious.	Sample preparation is comparatively easy
Acceleration voltage of 60 to 300KV is required to fire electrons. Resolution is higher than SEM	Acceleration voltage used is around 30KV. Resolution is comparatively lower than TEM
TEM can resolve any structure as small as 0.1 nm (including atomic level resolution)	SEM can easily resolve structures of size as low as 1 nm
Normally a 2-Dimensional image appears on a fluorescent screen but in advanced TEM, 3D image can also be seen	A 3D Picture appears on the Monitor

Sample preparation in Transmission Electron Microscopy (TEM)

It is a long and tedious process to prepare a sample (thin sections) for TEM. Each step requires precision and attention to preserve the integrity of the biological sample. Following are the key steps:

1. Fixation:

This step is important to preserve, immobilize and stabilize structure of biological specimen preventing their degradation. Common fixatives include aldehydes e.g., glutaraldehyde or formaldehyde.

2. Dehydration:

Dehydration of fixed sample can be done by passing it through a series of alcohol or acetone solutions of increasing concentration. It prepares the specimen for embedding.

3. Embedding:

The dehydrated specimen is embedded or inserted in an epoxy resin to provide support and stability to the delicate biological specimen for creation of ultra-thin sections.

4. Ultrathin Sectioning:

A diamond or glass knife fixed on a ultramicrotome is used to cut ultrathin sections (about 50-100 nm thick) of sample, embedded in epoxy resin block. These sections are then transferred on a support grid.

5. Staining:

Uranyl acetate and phosphotungstic acid acts as Negative stains to highlight specific features. These heavy metal negative stains, are repelled by the negatively charged cells and their components. As a result, the stains do not penetrate but surround the specimen, enhancing the electron scattering properties on the specimen's outer layers creating a dark background against which the specimen appears light. The specimen itself remains unstained. This creates a halo-like effect around the specimen.

6. Mounting:

The ultrathin sections are mounted on thin, flat support made of copper or gold. These materials are electron-dense and compatible with the high-vacuum environment of the TEM. Ultrafine forceps, are often used to transfer the sections on to the grid.

7. Drying:

The specimen is allowed to air dry at room temperature. This is a straightforward and less complex method but air drying can lead to shrinkage or distortion of specimens due to the evaporation of water, potentially introducing artifacts. An alternate method can be used i.e., Critical Point Drying (CPD) In which liquid within the sample is replaced with liquid carbon dioxide. The pressure and temperature adjusted to reach the critical point, where the liquid and gas phases coexist which can be removed gently to dry the sample.

The prepared TEM grid is loaded into the TEM instrument, and the specimen is observed under the electron beam.

Simplified TEM diagram

Components of TEM and their working

1. Microscope column:

The microscope column in Transmission Electron Microscopy (TEM) contains various elements such as Electron Gun, different lenses (condenser, objective, intermediate and projector lenses) and specimen holder. The column remains fitted with a Vacuum system composed of three types of vacuum pumps (roughing pump, diffusion or turbo pumps and ion pump) for maintaining a high vacuum within the column to prevent electron scattering.

Choice of materials for constructing the vacuum tube itself is crucial. Materials with low reactivity or low affinity for electrons are preferred. Common choices include glass for certain types of tubes and metals for others, Glass or metal surfaces (stainless steel or Nickel alloy or Titanium) are common choices because they have minimal interaction with electrons.

Materials with low atomic number are preferred to minimize electron scattering. The higher the atomic number, the more electrons there are in the atom. When incident electrons encounter atoms with higher atomic numbers, there is a greater chance of interactions, including scattering.

For coating the inner surface of vacuum tube, various materials and coatings can be employed for this purpose, and the choice depends on factors such as the type of vacuum tube. Internal surfaces coated with graphite or other conductive materials.

Some Anti-reflection coatings are designed to reduce reflections and increase the transmission of electrons through surfaces such as carbon, graphene or gold. These coatings can act as barriers, preventing electrons from being absorbed into the material of the tube.

Engineers carefully select and optimize coatings to minimize unwanted reflections and enhance the overall performance of electron microscopy

2. Electron Gun

Scanning is done by electrons, released by electron gun. The two main types of electron guns for TEM are:

· Thermionic Electron Gun: It is equipped either with Tungsten wire filaments or Solid-state hexaboride crystals of cerium hexaboride (CeB₆) or lanthanum hexaboride (LaB₆). These crystals are known for their stability, long life and high electron emission, releasing 10 times more electrons than tungsten.

· Field emission guns (FEG) uses fine tip (single crystal of tungsten) of 100 nm with a strong electrostatic field around the tip which provides a very small diameter electron beam. This ensures release of bright and stable beam to hit the specimen.

Two main types of electron guns for TEM

3. Anode:

The electrons emitted from the cathode (the Electron Gun) moves towards the positively charged anode. The anode creates an electric field that is crucial for accelerating the emitted electrons, leading to the generation of a high-energy electron beam. This process contributes to the shaping and focusing of this beam towards the specimen. The anode is composed of conductive material tungsten having a cylindrical or conical structure which can withstand high temperatures and impact caused by accelerated electrons.

4. Condenser lenses

There may be more than one condenser lenses to receive and focus the electron beam toward the specimen. Unlike SEM, which converges a pointed electron beam onto the specimen, the condenser lens in TEM focuses a broad beam at the sample. Additionally, a condenser aperture controls the size and angle of the electron beam thus controlling the amount of illumination reaching the specimen.

5. Objective lens:

Condenser is followed by objective lens. There are two objective lenses in TEM, one above and other below the specimen. The distance between the specimen and objective lens remains short with minimum focal length which gives maximum magnification and generate high resolution image. Upper objective lens along with condenser lens is mainly helps in parallel electron beam formation. The lower objective lens which positioned after the specimen receives the electrons transmitted or coming out from the specimen and responsible for image formation. It plays a crucial role in magnifying the image of the specimen by focusing the transmitted electrons.

6. Specimen holder:

The specimen holder is made up of metals or alloys that are electron-transparent to minimize interference with the electron beam. Advanced specimen holders may include a tilting mechanism, allowing researchers to tilt the specimen at various angles for studying the three-dimensional structure of specimens. The specimen should be extremely thin i.e., less than 100 nm because thick specimen may cause chromatic aberration due to inelastic electron scattering.

7. Intermediate lens:

It usually lies between the lower objective and the projector lens. Intermediate lens is typically a magnetic lens; by altering position of this lens and strength of its magnetic field observer can fine-tune the electron beam (controlling its convergence or divergence) which provides additional control over focus and magnification. This capability is valuable for obtaining detailed images at different scales. Aberrations, such as spherical and chromatic aberrations, can distort the image, and the intermediate lens also helps in correcting these distortions.

8. Projector lens:

The projector lens works in conjunction with the objective and intermediate lenses to project a focused electron beam, ensuring the provision of a magnified, sharp, and well-defined image onto the viewing screen or camera. The projector lens contributes to collimating (paralleling) the electron beam, ensuring its focus either on the fluorescent screen (for direct viewing by the observer) or onto the CCD (charge coupled device) camera for recording purpose.

9. Detectors:

TEM use different detectors to detect various signals for providing detailed information about the specimen.

Fluorescent Screen: The electrons when hit screen it fluoresces and emits visible light allowing researchers to directly view the specimen.

Photographic Film and CCD Camera: Photographic film records the pattern of transmitted electrons, while a CCD camera converts the pattern into electronic signals and form the digital image formation.

Electron Energy Loss Spectrometer(EELS):

When electrons pass through the specimen, they lose energy and this energy loss is measured by spectrometer providing information about the specimen's composition.

Digital Image of Virus with internal structures and its proteins spikes

Advantages of TEM

1. Cellular structures and associated pathologies: High resolution images of cellular and subcellular structures helps to understand the intricacies of cellular processes, organelle functions and structural changes associated with conditions such as cancer, neurodegenerative disorders, and infectious diseases.

2. Drug Development in pharmaceutical research: Interactions between drugs and cellular components can be visualized to optimize drug formulations for effective treatments with minimal side effects.

3. Developing vaccines and antiviral drugs: TEM is instrumental in Viral Research and defining the infections by understanding the virus structure, arrangement of protein receptors/spikes at the surface and mechanism of its invasion & replication inside host cells.

4. Reason for advancements in various fields: TEM provides diverse knowledge and understandings in the field of immunology, microbiology and genetics which is being widely used in biomedical research in accurately diagnosing certain diseases and conditions.

5. Nano-Medicine research: This advanced microscopy is helping to visualize and characterize nanoparticles for developing targeted drug delivery systems, and other nano-scale interventions in medicine.