Table of Contents
In this electron microscope principle construction and working post we have briefly explained about types of electron microscope technique, electron and sample interactions, instrumentation of SEM and TEM.
Electron Microscope Technique
The ultra-structure of cellular components such as the nucleus, plasma membrane, mitochondria, and others demands magnification of 10,000X or more, which Light Microscopes could not provide. Electron microscope technique, which have a stronger resolving power than light microscopes and can achieve higher magnifications, are used to do this.
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. As a result, compared to optical microscopes, electron microscopes provide a more detailed and crisp image.
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).
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.
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. By mounting samples on an aluminum stub, samples can be photographed directly in SEM.
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 utilised. 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 unscattered electrons through the specimen creates a picture.
Scanning electron microscope technique (SEMs) primarily utilise 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.
Electron Microscope Principle Construction and Working
Instrumentation of TEM
The optics of the TEM is similar to conventional transmission light microscope. A transmission electron microscope technique has the following components,
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. Unscattered 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.
Topography: surface features, texture
Morphology: shape and size of the particles Crystallographic arrangement of atoms
Composition: elements and the relative amounts.
Instrumentation of SEM
Knoll built it for the first time in 1935. It’s utilised to look at three-dimensional photographs of cell, tissue, or particle surfaces. The SEM enables for non-sectioned examination of specimen surfaces.
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.
SEMs have a detecting mechanism called an energy dispersive spectrometer (EDS) that can detect and display the majority of the X-ray spectrum. Semiconducting silicon or germanium is commonly used in detectors.
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.
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