Electron Microscopes�

By

SALONI SHARMA

Electron microscopes

• Electron microscopes- have a stronger resolving power than light microscopes and can achieve higher magnifications. Hence they are used to study the ultra-structure of cellular components such as the nucleus, plasma membrane, mitochondria, and others that demands magnification of 10,000X or more, which Light Microscopes could not provide.
• In an electron microscope technique, instead of light, a focused electron beam is utilized to study objects. When compared to the 400–700 nm wavelength of visible light used in an optical microscope, electrons are believed to be radiation with a wavelength in the range 0.001–0.01 nm.
• Optical microscopes have a maximum magnification power of 1000 times and a resolution of 0.2 m, but electron microscope technique have a resolving power of 1,000,000 times and a resolution of 0.2 nm

�Types of Electron Microscope�

• The electron microscope technique was invented in 1931 by two German scientists, Ernst Ruska and Max Knoll. Ernst Ruska later received Nobel Prize for his work in 1986. The Transmission Electron Microscope (TEM) was the first type of electron microscope technique to be developed.
• Transmission electron microscopes (TEM)
• Scanning electron microscopes (SEM)
• Scanning transmission electron microscopes (STEM).

​

�Principle�

• Electron microscope technique works on the same principles as a light microscope. Instead of photons, an electron microscope technique uses a high-velocity electron beam. Electrons are emitted from the cathode’s surface and propelled towards the anode by a high voltage to generate a high-energy electron stream in an electron gun.

​

• Electromagnetic lenses are used in the electron microscope technique. Magnetic fields interact with charged electrons, and the magnetic force focuses an electron beam. The beam diameter and convergence angles of the beam incident on a specimen are controlled by the condenser lens system. Either the transmitted beam or the diffracted beam is used to create the image. The picture is enlarged and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor.

Sample Preparation

• The most difficult and skilled phase in electron microscope technique is specimen preparation. The material to be examined using electron microscopy must be highly kept, fixed, thoroughly dried, ultrathin, and impregnated with heavy metals that intensify the distinction between diverse organelles.
• Fixation with glutaraldehyde and then osmium tetroxide are used to preserve the material. The fixed material is dehydrated before being implanted in plastic (epoxy resin) and sectioned with an ultra-diamond microtome’s or glass razor.
• Sample sections in TEM are ultrathin, measuring 50–100 nm in thickness. These sections are exposed to electron dense compounds such as lead acetate, uranylacetate, and phosphotungstate on a copper grid.
• In SEM the samples are mounted on an aluminum stub and can be photographed directly.

​

�Electron and Sample Interactions�

• Elastic scattered electrons, inelastic scattered electrons, secondary electrons, and backscattered electrons are all produced when an electron beam interacts with a substance.
• To acquire information about the material, almost any type of electron interaction can be utilized.
• Different aspects of the material, such as topography and elemental composition, can be determined depending on the type of radiation or released electrons employed for detection.

​

• A stream of electrons is transmitted through an ultrathin specimen in a transmission electron microscope technique (TEM), interacting with the material as it passes through. The interaction of the transmitted un-scattered electrons through the specimen creates a picture.
• Scanning electron microscope technique (SEMs) primarily utilize secondary electrons to image the surface topography of biological material. Secondary electrons and backscattered electrons are produced when an electron beam interacts with a material, which can be detected using typical SEM equipment.

​

�Instrumentation of TEM�

• The optics of the TEM is similar to conventional transmission light microscope. A transmission electron microscope technique has the following components:
• Electron gun
• Condenser lens
• Specimen stage
• Objective lens and
• Projector lens

​

• When a high voltage electric current (50,000–100,000 volts) is applied to a tungsten filament or cathode, electrons are emitted. A high voltage is put between the electron source (cathode) and an anode plate, resulting in an electric field that accelerates the electrons.
• In the microscope column, the released electrons move through vacuum. Vacuum is required to prevent severe electron scattering by gases. The electrons are focused into a very tiny beam using electromagnetic condenser lenses. After that, the electron beam passes through the specimen before passing via the electromagnetic objective lenses.
• The sample is in the middle of the column in a TEM microscope. Un-scattered electrons hit the fluorescent screen at the bottom of the microscope, creating an image of the specimen with its various sections displayed in varying degrees of darkness depending on their density.
• The image can be examined directly, photographed, or captured digitally.

​

Scanning Electron Microscope

• Scanning Electron Microscope (SEM) is a type of electron microscope that scans surfaces of microorganisms that uses a beam of electrons moving at low energy to focus and scan specimens
• The first Scanning Electron Microscope was initially made by Mafred von Ardenne in 1937 with an aim to:
• Surpass the transmission electron Microscope. He used high-resolution power to scan a small raster using a beam of electrons that were focused on the raster.
• Reduce the problems of chromatic aberrations images produced by the Transmission electron Microscopes.
• It is utilized to look at three-dimensional photographs of cell, tissue, or particle surfaces. The SEM enables for non-sectioned examination of specimen surfaces.

​

M. von Ardenne's first SEM

�Principle of Scanning Electron Microscope (SEM)�

• The Scanning electron microscope works on the principle of applying kinetic energy to produce signals on the interaction of the electrons.
• These electrons are secondary electrons, backscattered electrons, and diffracted backscattered electrons which are used to view crystallized elements and photons.
• Secondary and backscattered electrons are used to produce an image. The secondary electrons are emitted from the specimen and play the primary role of detecting the morphology and topography of the specimen while the backscattered electrons show contrast in the composition of the elements of the specimen.

​

Electron–matter interaction volume and types of signal generated

PARTS OF SEM

• The specimen is dehydrated in alcohol at 70°C after being fixed in liquid propane at 180°C. The dried specimen is then evaporated in a vacuum with a thin covering of a heavy metal, such as platinum or gold, to create an electron-reflecting surface.
• Samples are placed at the bottom of the electron column in SEMs, and electron detectors capture scattered electrons (backscattered or secondary). Several electromagnetic lenses, including condenser lenses and one objective lens, are used in SEM. Electromagnetic lenses are used to create electron probes rather than images, as in TEM.
• The electron beam’s crossing diameter is reduced by two condenser lenses. The electron beam’s cross-section is further reduced by the objective lens, which focuses the electron beam as a probe on the specimen surface. As a result, the objective lens acts as a condenser. This is in contrast to TEM, which uses an objective lens to magnify the image.

​

• The major components of the Scanning Electron Microscope include;
• Electron Source – This is where electrons are produced under thermal heat at a voltage of 1-40kV. the electrons condense into a beam that is used for the creation of an image and analysis. There are three types of electron sources that can be used i. e Tungsten filament, Lanthanum hexaboride, and Field emission gun (FEG)
• Lenses – it has several condenser lenses that focus the beam of electrons from the source through the column forming a narrow beam of electrons that form a spot called a spot size.
• Scanning Coil – they are used to deflect the beam over the specimen surface.
• Detector – It’s made up of several detectors that are able to differentiate the secondary electrons, backscattered electrons, and diffracted backscattered electrons. The functioning of the detectors highly depends on the voltage speed, the density of the specimen.
• The display device (data output devices)
• Power supply
• Vacuum system
• Like the transmission electron Microscope, the Scanning electron microscope should be free from vibrations and any electromagnetic elements.

​

• The source of the electrons and the electromagnetic lenses are from tungsten filament lamps that are placed at the top of the column and it is similar to those of the transmission electron Microscope.
• The electrons are emitted after thermal energy is applied to the electron source and allowed to move in a fast motion to the anode, which has a positive charge.
• The beam of electrons activates the emission of primary scattered (Primary) electrons at high energy levels and secondary electrons at low-energy levels from the specimen surface. The beam of electrons interacts with the specimen to produce signals that give information about the surface topography and composition of the specimen.
• The specimen does not need special treatment for visualization under the SEM, even air-dried samples can be examined directly. However, microbial specimens need fixation, dehydration, and drying in order to maintain the structural features of the cells and to prevent collapsing of the cells when exposed to the high vacuum of the microscope.
• The samples are mounted and coated with thin layer of heavy metal elements to allow spatial scattering of electric charges on the surface of the specimen allowing better image production, with high clarity.

​

​

• Scanning by this microscope is attained by tapering a beam of electrons back and forth over a thin section of the microscope. When the electrons reach the specimen, the surface releases a tiny staw of electrons known as secondary electrons which are then trapped by a special detector apparatus.
• When the secondary electrons reach and enter the detector, they strike a scintillator (a luminescence material that fluoresces when struck by a charged particle or high-energy photon). This emits flashes of light which get converted into an electric current by a photomultiplier, sending a signal to the cathode ray tube. This produces an image that looks like a television picture that can be viewed and photographed.
• The quantity of secondary electrons that enter the detector is highly defined by the nature of the specimen i.e raised surfaces to receive high quantities of electrons, entering the detector while depressed surfaces have fewer electrons reaching the surface and hence fewer electrons enter the detector.
• Therefore raised surfaces will appear brighter on the screen while depressed surfaces appear darker.

​

Working of the SEM

• The working of a scanning electron microscope typically depends on the detection of the reflected electrons after they strike the surface of a specimen. The main element of a scanning electron microscope is an electron source. Generally, the heated tungsten filament is used as the electron source in most scanning electron microscopes. Here, the heat tends to supply more energy to the electrons, thereby directing them in a particular direction and producing a single focused electron beam. An anode or a positively charged electrode plate is present between the electron source and the condenser. The main purpose of the anode is to deflect the electrons away and align them in a thin and single straight line. This is because the electrons contain a negative charge on them, while the anode plate is positively charged. A scan coil and an objective lens are present below the condenser. The electron beam generated by the source passes through the condenser, scan coil, and objective lens. 

• The electron beam generated by the source passes through the condenser, scan coil, and objective lens. When the electrons contained by the electron beam hit the sample, they reflect and get scattered in all directions randomly. This is known as electron escape, which helps the user to establish a relationship between the number of scattered and retained electrons.
• The signal that is produced as a result of the electron-sample interaction and electron escape gets detected by the detector. The detector is further connected to a sensor. The sample generally consists of bumps and valleys.
• When the electrons hit the bump area of the sample, a greater number of electrons tend to escape, and when the electrons hit the valleys, comparatively few electrons manage to reflect and escape. This difference in the escape of the electrons helps to develop an image of the sample at the micro-level.

�Applications of the SEM�

• A scanning electron microscope is used as an analysis tool in a number of fields including biology, pharmaceuticals, manufacturing industries, physics laboratories, and many more. Some of the prime uses of the scanning electron microscope are listed as follows:
• Scanning electron microscope is widely used in energy-Dispersive X-ray Spectroscopy for spot chemical analysis.
• It is prominently employed in biology laboratories to study the internal structures of microorganisms at the cellular level.
• A scanning electron microscope has multiple applications in industries. For instance, it can be used to study the surface of solid objects and analyze the distribution of atoms in various elements.

​

•  Cosmetologists make use of a scanning electron microscope to analyze fine details of tiny cosmetic components.
• Manufacturing industries employ scanning electron microscopes to look for the contaminants and impurities in manufactured items.
• Quality control departments of various industries use scanning electron microscopes to determine the purity of a particular substance. For instance, pharmaceutical industries use them for good-bad testing of drugs, medicines, and other products.
• Scanning electron microscope is also used in qualitative chemical analysis of elements by proving a clear and magnified image of the crystalline structures.
• A scanning electron microscope is quite advantageous in nanotechnology and other related fields. It can provide precise measurements and detailed images of objects that are over 50nm in size.

​

• It can be used to distinguish different phases of a multiphase sample.
• Some of the scanning electron microscopes are equipped with diffracted backscattered electron detectors, which helps to examine and determine the micro-fabric and crystallographic orientation of substances.
• A scanning electron microscope is typically used to produce high definition images of objects that can display spatial variation of chemical compounds.
• A scanning electron microscope is generally preferred when it is required to perform the analysis of selective point locations on the sample.
• It is generally used in the medicine domain to observe the bacterial interaction with the skin and body organs. This helps the doctors determine the nature of the bacterial disease and to find the cure.

​

�Advantages of the SEM�

• Scanning electron microscopes are user friendly and easy to use.
• They can produce and generate results in digital format.
• Scanning electron microscopes are able to provide quick results, i.e., data can be obtained within a few minutes.
• A scanning electron microscope requires minimum sample preparation.
• The resolution of scanning electron microscopes is significantly high.

​

�Disadvantages of SEM�

• Scanning electron microscopes are comparatively expensive.
• 2. Some microscopes must fulfil certain special conditions before their use. For instance, the room must be free of vibrations and electromagnetic radiation.
• 3. Scanning electron microscopes have a bulky structure.
• A consistent voltage level must be maintained for the proper operation of a scanning electron microscope. This may require additional electronic circuitry or voltage regulators to fix the voltage magnitude to a constant value.

• A cooling system should be attached with such microscopes.
• The sample should be small enough to fit within the chamber of the microscope. The horizontal dimensions of the sample should not exceed 10 cm, while the vertical dimension is much more constrained and must be less than 40 mm.
• The sample to be examined with the help of a scanning electron microscope needs to be solid in nature. Wet samples are unsuitable and are required to be decrepitated first.
• A scanning electron microscope can not be used for light materials such as hydrogen, helium, lithium, etc.
• To study samples that are insulators with the help of a scanning electron microscope, an electrically conductive coating is required to be applied on their surface. However, it can be neglected if the device is able to work in low vacuum mode.
• Scanning of living samples with the help of a scanning electron microscope is not possible.

​

​

Scanning transmission electron microscopy (STEM)

• Scanning transmission electron microscope technique (STEM) is a technique that combines the principles of transmission and scanning electron microscope technique and can be done on any type of apparatus. STEM, like TEM, necessitates extremely thin samples and focuses on beam electrons transmitted by the sample.
• One of its main advantages over TEM is that it allows the utilisation of signals such as secondary electrons, scattered beam electrons, distinctive X-rays, and electron energy loss that cannot be spatially correlated in TEM.

Difference between SEM and TEM

THANK YOU