What is Electron Microscopy?
Electron microscopy (EM) is a technique to obtain images of biological and non-biological specimens with greater resolution.
It is utilized in biomedical exploration to examine the nitty gritty design of tissues, cells, organelles and macromolecular complexes.
The high resolution of EM pictures results from the utilization of electrons as the origin of illuminating radiation.
EM is utilized simultaneously with ancillary techniques like thin sectioning, negative staining etc. to address explicit questions.
EM pictures give key detail on the basic structure of cell function and of cell disease.
Electron Microscopy Principle
An electron microscope utilizes an ‘electron beam’ to create the pictorial representation of the object and magnification is gotten by ‘electromagnetic fields’; in contrast to light or optical microscopes, in which ‘light waves’ are utilized to deliver the picture and magnification is acquired by an arrangement of ‘optical focal lenses’.
It has effectively been examined that, the more modest is the frequency of light, the more noteworthy is its resolving power.
The frequency of green light (=0.55µ) is 1, 10,000 times longer than that of electron beam (=0.000005µ or 0.05 Å; 1µ = 10,000 Å).
That is the reason, in spite of its more modest mathematical aperture, an electron microscope can resolve objects as little as 0.001µ (=10 Å), when contrasted with 0.2µ by a light microscope.
Subsequently, as compared to light microscope, the resolving power of an electron microscope is 200 times more prominent.
For instance, if light microscope produces magnification up to ×2000, then electron microscope produces up to ×400,000 magnification.
Types of Electron Microscope
There are two important types of electron microscope – the transmission EM (TEM) and the scanning EM (SEM).
I. Transmission Electron Microscope (TEM)
The transmission electron microscope (TEM) is utilized to observe thin specimens (tissue sections, molecules, etc.) through which electrons can pass producing a projection image.
The TEM does closely resemble in various angles with the conventional (compound) light microscope.
TEM is utilized, in addition to other things, to image the inside of cells, the construction of protein molecules (differentiated by metal shadowing), the association of molecules in infections and cytoskeletal filaments (ready by the negative staining technique), and the course of action of protein molecules in cell membranes (by freeze-fracture).
II. Scanning Electron Microscopy
Scanning electron microscopy relies upon the emission of secondary electrons from the surface of a specimen.
As a result of its incredible profundity of focus, a scanning electron microscope is the EM simple of a stereo light microscope.
It gives detailed pictures of the surfaces of cells and entire living beings that are not possible by TEM.
It can likewise be utilized for particle counting and size determination, and for measure control.
It is named a scanning electron microscope on the grounds that the picture is framed by scanning a focused electron beam onto the surface of the specimen in a raster pattern.
The interaction of the primary electron beam with the atoms close to the surface result in the emission of particles at each point in the raster.
These can be gathered with an assortment of detectors, and their overall number meant brilliance at every equivalent point on a cathode beam tube.
Since the size of the raster at the specimen is a minute than the survey screen of the CRT, the resulting picture is a magnified image of the specimen.
Fittingly prepared SEMs (with secondary, backscatter and X-beam detectors) can be utilized to analyse the topography and atomic distribution of specimens, and furthermore, for instance, the surface circulation of immuno-labels.
III. Reflection Electron Microscope (REM)
Likewise, TEM in REM, an electron beam is incident on a surface yet rather than utilizing the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is distinguished.
This technique is normally combined with reflection high-energy loss spectroscopy (RHELS) and reflection high energy electron diffraction (RHEED).
In addition to this, another variety is spin-polarized low-energy electron microscopy (SPLEEM), which is utilized for taking a gander at the microstructure of magnetic spaces.
IV. Scanning Transmission Electron Microscope (STEM)
The STEM rasters a focused incident probe all around a specimen that (likewise with the TEM) has been thinned to enable identification of electrons scattered through the specimen.
The high resolution of the TEM is subsequently conceivable in STEM.
The focusing activity (and distortions) happen before the electrons bombard the specimen in the STEM, yet thereafter in the TEM.
The STEMs utilization of SEM-like beam rastering works on annular dark-field imaging, and other logical techniques, yet in addition implies that picture information is obtained in serial instead of in parallel fashion.
Regularly TEM can be outfitted with the scanning option and afterward it can work both as TEM and STEM.
V. Scanning Tunneling Microscopy (STM)
In STM, at high voltage a conductive tip held is brought close to a surface, and a profile can be received depending on the tunneling probability of an electron from the tip to the example since it is an function of distance.
Disadvantages of Electron Microscopy
Electron microscopes are extravagant to assemble and keep up with, yet the funds and running expenses of confocal light microscope system currently covers with those of fundamental electron microscopes.
Microscopes intended to accomplish high resolutions should be housed in stable structures with uncommon administrations, for example, magnetic field cancelling systems.
The examples to a great extent must be seen in vacuum, as the molecules that make up air would scatter the electrons.
An exemption is liquid-phase electron microscopy utilizing either a closed liquid cell or environmental chamber, for instance, in the environmental scanning electron microscope, which permits hydrated examples to be seen in a low-pressure (up to 20 Torr or 2.7 kPa) wet environment.
For in situ electron microscopy of different techniques, gaseous samples have also been created.
SEM working in traditional high-vacuum mode normally image conductive specimens; in this way non-conductive materials require conductive covering (gold/palladium combination, carbon, osmium, and so forth).
It becomes possible to observe a non-conductive material without coating using the low-voltage mode of modern microscopes.
Minute, stable specimens like carbon nanotubes, diatom frustules and little mineral crystals (asbestos filaments, for instance) doesn’t require any special treatment prior being observed in the electron microscope.
Samples of hydrated materials, including practically all biological specimens must be ready in different manners to balance out them, diminish their thickness (ultrathin sectioning) and increases their electron optical contrast (staining).
These cycles might bring about artefacts, however these can for the most part be recognized by contrasting the outcomes got by utilizing drastically unique specimen arrangement methods.
Since the 1980s, examination of cryofixed, vitrified specimens has additionally gotten progressively utilized by researchers, further affirming the legitimacy of this technique.
Electron Microscopy Citations
- A primer on resolving the nanoscale structure of the plasma membrane with light and electron microscopy. J Gen Physiol . 2019 Aug 5;151(8):974-985.
- Electron microscopy of specimens in liquid. Nat Nanotechnol . 2011 Oct 23;6(11):695-704.
- Diagnostic Electron Microscopy of Viruses With Low-voltage Electron Microscopes. J Histochem Cytochem . 2020 Jun;68(6):389-402.
- Quantitative Cryo-Scanning Transmission Electron Microscopy of Biological Materials. Adv Mater . 2018 Oct;30(41):e1706681.