What is transmission electron microscope?
- In a Transmission electron microscope, the electron beam is transmitted through a very thin specimen or object and forms a highly magnified and detailed image of the sample.
- This microscope uses electron beams instead of light.
- The specimen used in Transmission Electron Microscope, should be very thin, less than 100 nm thick.
- A Transmission Electron Microscope can create a much higher resolution and magnified image than a light microscope, because of the shorter wavelength of the electron as compared to photons.
- In TEM the sample’s image is formed by the interaction between the transmitted electron beam and sample.
- TEM can tell us the structure, crystallization, morphology, and stress of the specimen in a better way as compared to a simple microscope.
- The formed image is then magnified and visualized on a fluorescent screen (layer of photographic film).
- Ernst Ruska and Max Knolls discovered the first Transmission Electron Microscope in 1931.
Parts of A Transmission Electron Microscope
1. Electron Gun
- Electron guns consist of four important parts the filament, a biasing circuit, a Wehnelt cap, and an extraction anode.
- When an electron gun is connected with a power supply it starts to generate electron beams.
- These electron beams are now moved towards the anode plate and the TEM column.
- It generates electron beams.
2. Vacuum system
It creates a vacuum to prevent the interaction between air particles and electrons. So that electron will not be scattered.
3. Specimen stage
It has an airlocks system to insert the specimen inside the vacuum with minimal loss of vacuum in other areas of the microscope.
4. Electron lens
These act as an optical lens by focusing parallel electrons at some constant focal distance.
- Apertures are annular metallic plates, which consist of a small metallic disc.
- This disc permits the axial electrons to pass through it and exclude those electrons that are at a distance from the optic axis.
- By permitting the central electrons, apertures decrease the intensity of electron beams in TEM, which is good for the beam sensitive samples.
- By this way it also removes those electrons are scattered to high angles.
6. Image-Recording System
- It consists of the fluorescent screen that is used to view the image and the focus device. A digital camera is also included that records images after viewing.
- A vacuum system prevents electrons from colliding with air molecules, causing them to move and focus differently. Vacuuming allows electrons to move straight to the image.
- The vacuumed system consists of a pump and gauge, valves, and a power supply.
- Monochromatic images are greyish, black and white. The electrons must be visible to the eye. Therefore, they are allowed to pass through the fluorescent screen at the base.
- Digitally, the image can be taken and displayed on a computer. The JPEG and TIFF formats can also be saved. The image can be transformed from its monochromatic state into a colored one depending on what recording device is used. For example, pixel cameras can store an image in color.
- Colored images allow for easy identification and characterization of images.
Working principle of Transmission Electron Microscope
Electron Microscope follows the same principle as a light microscope follow. The major difference is, the light microscope uses artificial light or natural light to create an image of the specimen, whereas an electron microscope uses electron beams.
In EM when electron beams cross through a specimen, the electron particles are started to scatter. The electromagnetic lens on EM focusses the scatter electron on a screen and creates an image of the specimen.
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How does the transmission electron microscope work?
- First of all, a tungsten filament is heated, which is also called an electron gun.
- The heated tungsten filament or electron gun will start to release electron beams.
- An electromagnetic coil and high voltage(up to several million volts) applied to these electron beams to accelerate their speed (extremely high speeds ).
- A condenser lens with a high aperture eliminates all the high angle electrons and focused all the electron beams into a thin, small beam.
- The high-speed electron beams are now transmitted through the specimen.
- The transmitted electron beams are focused into an image with the help of an objective lens.
- The vaccine chamber of TEM prevents the collide of electrons with the gas atoms.
- The electron beams are projected on to a phosphorescent screen, which creates an image of the specimen, also called a micrograph.
- All the images are captured by a charge-coupled device (CCD) camera, which is located underneath the screen.
Sample Preparation for TEM
It is a very complex process. The specimen should be less than 100 nanometers thick, otherwise, electron beams can not pass through it. The specimen should be 20-100nm thin and 0.025-0.1nm diameter.
Sample preparation for TEM is accomplished in the following steps;
1. Fixation of Specimen:
It helps to stabilize the cells, which prevents further change or damage to the cells. This can be done in two methods;
a. Chemical fixation of specimen:
It helps to stabilize the biological samples. The cross-linking of protein molecules with nearby molecules is done by those Chemical substances. Glutaraldehyde is mostly used in this method.
b. Cryofixation fixation of specimen:
In this method, the specimen is a dip into liquid nitrogen or liquid helium for rapid freezing. It will help to form a vitreous ice from the water content of the sample.
- The sample is rinsed properly using a buffer such as sodium cacodylate to prevent the increase of acidity in the sample.
- It will maintain the pH in the specimen.
3. Secondary fixation
- It is carried out with the help of osmium tetroxide (OsO4).
- Secondary fixation helps to increase the contrast of the tiny structures inside the specimen and provides more stability.
- This converts the proteins into gels, as a result, which increases the contrast between nearby cytoplasm by binding regions of phospholipid heads.
- This method involves the replacement of water content in the specimen with an organic solvent such as Ethanol and acetone.
- This process prevents the epoxy resin to mix with the water content.
- In this method epoxy resin is added to the specimen, it will occupy all the space in the cell.
- It will make the spacemen too hard that it can bear the pressure of sectioning or cutting, this method also called embedding.
- Now the resin is kept in an oven at 60° overnight to allow for setting, this method is called polymerization
- This is done with the Ultrafine abrasives, to give the sample a mirror-like finish.
- This step helps to reduce the scratches on the sample and also minimize other problems that can reduce the quality of the image.
- The main purpose of this step is to make the specimen semi-transparent, which will allow the electron beams to pass through it.
- This step is done with a device called an ultramicrotome.
- Ultramicrotome contains a glass or diamond knife, which cuts the specimen into finesections (30 nm and 60 nm ).
- After this step, the specimen is moved to a copper grid to be viewed under the microscope.
- The staining of the specimen is done into an aqueous solution of heavy metals like uranium, lead, or tungsten.
- It will help to increase the contrast between different structures in the specimen, and also to scatter the electron beams.
*The preparation of cryofixed specimen is done by directly cutting them and then shadowed using vapors of platinum, gold, or carbon before visualization under the TEM.
The preparation of the specimen can also be done by the following methods;
- Cross-sectional method
- Replica technique
- Electrolyte polishing
What is Freeze-itching treatment?
Freeze-itching is used to treat microbial cells in order to reduce the potential dangers associated with artifacts. This is unlike chemical fixation and dehydration which can lead to contamination.
- A special treatment for microbial cell organelles is Freeze-itching. The specimens are first prepared with liquid nitrogen, then heated in a vacuum chamber at -100°C.
- Next, the sections are cut using a precooled knife in liquid Nitrogen at -196°C. The sectioned specimen can be heated in high vacuum for approximately 2 minutes. After that, the platinum and carbon layers can be applied to it.
- These can then be viewed in the TEM, which displays more detailed internal structures of each cell in 3D.
- This is the freeze-itching stage of Liquid Nitrogen treatment.
Image Formation in TEM
- After releasing from the electron gun, a condenser lens focused the electron beam on to a specimen.
- Condenser aperture excludes high angle electrons from the electron beam.
- Now some of the electron beams strike the specimen and transmit through it.
- An objective lens focused this transmitted electron beam on a phosphorus screen or charge-coupled device (CCD) camera to form an image of the specimen.
- An optional objective aperture can be used to block the high-angle diffracted electrons to increase the contrast of the image.
- Now the image is passed through the column and projector lens to form an enlarged image.
- When the image strikes the phosphorus screen it generates light, allowing the viewer to see the image.
- The dark area of the image indicating those areas on the sample where fewer electrons were transmitted.
- The lighter area of the image indicating those areas on the sample where more electrons were transmitted.
What is Diffraction Point?
After passing through the specimen, the electrons are started to scattered due to the electrostatic potential set up by the constituent elements in the specimen.
The transmitted electrons are now passed through an electromagnetic objective lens which helps to focus all the scattered electrons from one point of the sample or specimen into one point in the image plane.
Also, shown below image is a dotted line where the electrons scattered in the same direction by the sample are collected into a single point. This is the back focal plane of the objective lens and is where the diffraction pattern is formed.
Application of Transmission Electron Microscope
- TEM is used to study the topographical, morphological, compositional, and crystalline information.
- It helps to analyze the structure and texture of the specimen.
- TEM used in semiconductor analysis.
- TEM is also used in production and the manufacturing of computers and silicon chips.
- It is also used in Technology companies to identify flaws, fractures, and damages to micro-sized objects.
- TEM is also used in Colleges and universities for research and study purposes.
- To distinguish between animal and plant cells.
- It is also used in nanotechnology for studying nanoparticles like ZnO nanoparticles
- It can be used to identify and detect fractures.
- To study and visualize the cell structures of bacteria, viruses and fungi.
- View bacteria flagella and plasmids
- View the sizes and shapes of microbial cell organelles
Advantages of Transmission Electron Microscope (TEM)
- TEM provides 1 million times of magnification power as compared to a simple microscope.
- It provides detailed information about the structure of specimens.
- TEM can produce a high-quality and detailed image of the specimen.
- It is easy to operate with proper training.
- Permanent images can be produced by it.
- It can be used in a wide variety of ways, including basic Biology and Nanotechnology, education, and industrial applications.
Limitations of Transmission Electron Microscope (TEM)
- They are very expensive.
- TEM is very large in size, they require a room to operate.
- Required proper training to prepare the specimen for TEM.
- It produces monochromatic pictures unless it uses a fluorescent screen at visualization’s end.
- They are sensitive to electromagnetic movements and vibrations, so they are only used in areas where they are protected.
- To function, it requires constant voltage flow.
- They can be difficult to maintain.
- Artifacts can be dangerously damaged by chemical fixations, dehydrators, or embedments.
- Only electron transparent specimens are used in TEM.
- To operate a TEM requires special training.
- It is tedious to prepare specimens for viewing under the TEM.
Transmission electron microscope images