INTRODUCTION:

· The electron microscope use electron beams and magnetic fields to produce the image instead of light waves and glass lenses used in the light microscopes.

· Resolving power of electron microscope is far greater than that of any other compound microscope.

· This is due to shorter wavelengths of electrons. The wavelength of electrons is about 100,000 times smaller than the wavelength of visible light.

· Microscope forms an enlarged image of the original object in order to convey its internal or external structure.¹ The magnification and resolution of microscope is given as -Magnification- objective lens x eyepiece.

· Resolution (r) is defined as closest spacing of two points which can be clearly seen through the microscope as separate entities. Resolution can be determined as:

R_d- 0.61λ = 0.61 λ/ NA

μ sinα

Where,

λ = wavelength of light

μ = refractive index of the medium between the object and objectives lens

α = the half angle made by aperture at the specimen

NA = μsinα =numerical aperture

By increasing the dimension or by employing large number of lenses, the magnification can be increased, while shorter wavelength yields higher resolution.²

Method for Electron Microscope:

· The specimen to be observed is prepared as extremely thin dry film on small screens.

· These are then introduced into the instrument at a point between the magnetic condenser and the magnetic objective.

· The magnified image is viewed on a fluorescent screen through an airtight window.

· The image can be recorded on a photographic plate by a camera built into the instrument.^{3, 4}

Types:

Mainly 2 types:

· Transmission Electron Microscope (TEM) - allows one the study of the inner structures.

· Scanning Electron Microscope (SEM) - used to visualize the surface of objects.

1. Transmission Electron microscope:

The optics of the TEM is similar to conventional transmission light microscope.⁵ It was developed in 1930s. Information that can be obtained using TEM includes:

· Topography: surface features, texture

· Morphology: shape and size of the particles

· Crystallographic arrangement of atoms

· Composition: elements and the their relative amounts

· It is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it.

· A transmission electron microscope can achieve better resolution and magnifications of up to about 10,000,000 xs.^6,7

Fig: Transmission Electron Microscope

Construction:

· Electron gun: It consists of a tungsten filament or cathode that emits electrons on receiving high voltage electric current (50,000-100,000 volts).

· Ray tube (Microscope Column): It is a high vacuum metal tube (2mt. high) through which electrons travel.

· Condense lens: It is the electromagnetic coil which focuses the electron beam in the plane of the specimen.⁸

· Objective lens: It is the electromagnetic coil which produces the first magnified image formed by the objective lens and produces the final image.

· Projector lens: It is also an electromagnetic coil which further magnifies the first image formed by the objective lens and produces the final image.

· Fluorescent Screen or Photographic Film: Since unaided human eye cannot observe electrons, therefore, a fluorescent screen is used for viewing the final image of the specimen.⁹

Interaction of electron beam with sample:

Interaction of electron beam with the sample result is three types of electrons:

1. Unscattered electrons: These are electrons that are transmitted through the thin specimen without any interaction with the sample. Transmission of unscattered electrons is inversely proportional to the specimen thickness. The areas of specimen that are thicker will have fewer transmitted unscattered electrons and will appear darker. Conversely thinner areas will have more transmitted electrons and will appear lighter.¹⁰

2. Elastic scattered electrons: These are incident electrons that are scattered by the atoms of the specimen in elastic fashion that is without any loss of energy of electrons. The elastically scattered electrons form diffraction pattern that yield information about the orientation, atomic arrangements and phases present in the area being examined.

3. Inelastic scattered electrons: Incident electrons that interact with the specimen atoms in inelastic fashion lose energy during the interaction fall in this catagory. The extent of loss of energy by incident electrons depends on the characteristic of interacting elements and is used to study the compositional and bonding (i.e. oxidation state) information’s of specimen region being examined.^11,12

Working principle and instrumentation:

An electron gun at the top of the microscope emits electrons that travel through vacuum in the microscope column Vacuum is essential to prevent strong scattering of electrons by gases. Electromagnetic condenser lenses focus the electrons into a very thin beam Electron beam then travels through the specimen and then through the electromagnetic objective lenses. At the bottom of the microscope, unscattered electrons hit the fluorescent screen giving image of specimen with its different parts displayed in varied darkness, According to their density. The image can be studied directly photographed or digitally recorded.¹³

Sample Preparation:

Sample preparation is important for electron microscopy. There are three main steps for sample preparation: Processing, embedding and polymerization.

Processing This includes: fixation, rinsing, post fixation, dehydration and infiltration.

Fixation: This is done to preserve the sample and to prevent further deterioration so that it appears as close as possible to the living state, although it is dead now. Eg. Gluteraldehyde fixation for proteins.

Rinsing: The samples should be washed with a buffer to maintain the pH. This prevents extra acidity.

Post fixation: A secondary fixation with osmium tetroxide (OsO4), which is to increase the stability and contrast of fine structure.

Dehydration: The water content in the tissue sample should be replaced with an organic solvent since the epoxy resin used in infiltration and embedding step are not miscible with water.

Infiltration: Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to give hard plastic material which will tolerate the pressure of cutting.^15,16

Embedding After processing the next step is embedding. This is done using flat molds.

Polymerization: Next is polymerization step in which the resin is allowed to set overnight at a temperature of 60 degree in an oven.

Sectioning: The specimen must be cut into very thin sections for electron microscopy so that the electrons are semitransparent to electrons.^17,18

Advantages:

· TEMs offer very powerful magnification and resolution.

· TEMs have a wide-range of applications and can be utilized in a variety of different scientific, educational and industrial fields

· TEMs provide information on element and compound structure.

· Images are high-quality and detailed.

Disadvantages:

· TEMs are large and very expensive.

· Laborious sample preparation.

· Operation and analysis requires special training.

· Samples are limited to those that are electron transparent.

· TEMs require special housing and maintenance.

· Images are black and white.^{19. 20}

2. Scanning Electron Microscope (SEM):

SEM is most widely used type of electron microscope for study of microscopic structure. In SEM, image is formed by focused electron beam that scans over the surface area of specimen. The incident beam in SEM is also called electron probe. The incident beam is of typically 10mm diameter in contrast to beam of TEM which is about 1 im. In SEM, image is not within the specimen or even adsorbed rather than transmitted. SEM overcomes this limitation formed by instantaneous illumination of the whole field as for TEM.^21,22

Principle of Sem- Accelerated electrons in an SEM carry significant amounts of kinetic energy, and this energy is dissipated as a variety of signals produced by electron-sample interactions when the incident electrons are decelerated in the solid sample. These signals include secondary electrons that produce SEM images.²³

Electron-Sample Interactions:

The interaction of electron beam with samples results in secondary electrons and backscattered electrons that are detected by standard SEM equipment Secondary Electrons: As incoming electrons pass through the specimen, they impart some of their energies to electrons of nearby specimen atom. This causes ionization of the electrons of the specimen atom and slight energy loss and path change of the incident electrons.^[24] These ionized electrons then leaves the atom with a very small kinetic energy (5eV) and are termed as secondary electrons. The secondary electrons escape from a volume near the specimen surface, at a depth of 5-50 nm and hence are useful to gain topography related informations.²⁵

Backscattered Electrons:

Some of the electrons of the incident beam collide with the specimen atoms that fall in the path and are reflected or back scattered. The production of backscattered electrons varies directly with the specimen's atomic number of the specimen. When backscattered electrons are detected, higher atomic number elements appear brighter than lower atomic number elements. This interaction is utilized to differentiate parts of the specimen that have different average atomic number.²⁶

Working:

· The electron gun produces an electron beam which is accelerated by the anode.

· The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample.²⁷

· A mechanism of deflection coils enables to guide the beam so that it scans the surface of the sample in a rectangular frame.

· When the beam touches the surface of the sample, it produces: – Secondary electrons (SE) – Back scattered electrons (BSE) – X - Rays.²⁸

· The emitted SE is collected by SED and converts it into signal that is sent to a screen which produces final image.

Advantages:

· It gives detailed 3D and topographical imaging and the versatile information garnered from different detectors.

· This instrument works very fast.

· Modern SEMs allow for the generation of data in digital form.

· Most SEM samples require minimal preparation actions.

Disadvantages:

· SEMs are expensive and large.

· Special training is required to operate an SEM.

· The preparation of samples can result in artifacts.

· SEMs are limited to solid samples.

· SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface.³⁹

Fig: Scanning Electron Microscope

CONCLUSION:

This article has sought to discuss scanning electron microscopy and transmission electron microscopy in terms of its principles, applications, and advantages with respect to other imaging systems. In the section on fundamentals and principles, a selection of pertinent literature was integrated into the discussion to provide a good introduction into this general field.

CONFLICT OF INTEREST:

The author declares that they have no conflict of interest.

REFERENCES:

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3. Lyman CE, Newbury DE, Goldstein JI, Williams DB, Romig AD, Armstrong JT, Echlin P, Fiori CE, Joy DC, Lifshin E, Peters KR, 1990 Scanning Electron Microscopy X-Ray Microanalysis and Analytical Electron Microscopy: A Laboratory Workbook, Press. New York, N.Y.

4. Postek MT, Howard KS, Johnson AH, McMichael KL. 1980. Scanning Electron Microscopy: A Student’s Handbook, (Ladd Research Ind., Inc. Williston, VT).

5. Eicixen E, Fitchmun DR, Sefton LR. 1969. Interpretation of micrographs from a scanning electron microscope.

6. Proc., 27th Ann. Meeting, Electron Microscopy Society of America (C. J. Arceneaux ed.). Claitors' Publishing Division, Baton Rouge, p. 22-23. Everhart, E. 1958. Contrast formation in the scanning electron microscope. Ph.D. Dissertation. University of Cambridge (England).

7. Smith A, Wells OC, Oatle CW, 1960. Recent developments in scanning electron microscopy. Vierter Internationater Kongress Fur Elektronen-mikroskopie (Herausgegeben von 6. Mollenstedt, H. Niehrs, and E. Ruska). Band I, p. 269- 273.

8. Springer-Verlag, Berlin (in English)., 0. C. Wells, C. W. Oatley 1959. Factors affecting contrast and resolution in the scanning electron microscope. J. Electronics and Control, 7(2): 97-111. Fengel, D. 1967. Ultramicrotomy, its applications in wood research. Wood Science and Technology, l(3) : 191-204.

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10. Forgacs 0L. 1963. The characterization of mechanical pulps. Pulp and Paper Magazine of Canada (Convention Issue), 64. (C): T89- T118.

11. Fujiyasu Tk, Tilwura, H. 1968. Hitachi scanning electron microscope. Proc., 26th Ann. Meeting, Electron Microscopy Society of America (C. J. Arceneaux, ed.). Clai- 132 Bernard M. Collett tors' Publishing Division, Baton Rouge, p. 374- 375.

12. Hall CE 1966. Introduction to electron microscopy. 2nd ed. McGraw-Hill Book Co., New York. 397 p.

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15. Jensen WA, Park RB 1967. Cell ultrastructure. Wadsworth Publishing Company, Inc., Belmont, California. 60 p. KAY, D. H. 1965.

16. Techniques for electron microscopy. 2nd ed. F. A. Davis Co., Philadelphia. 560 p. KIMOTO, S. 1967.

17. On a scanning electron microscope. Bulletin No. SM-67013. Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan. 15 p.

18. Philip D. Rack, on “Optical Microscopy”, Dept. of Materials Science and Engineering University of Tennessee & lecture was generated by Professor James Fitz-Gerald at the University of Virginia.

19. Gabriel Popescu, “Chapter 4. Principles of Optical Imaging” Electrical and Computer Engineering, University of Illinois at Urbana‐Champaign, Beckman Institute Quantitative Laboratory, Light Imaging http://light.ece.uiuc.edu

20. Text of Microbiology web sitehttps://www.ikbooks.com/ home/sample chapter? filename=147_Sample-Chapter.pdf 7.

21. Khangaonkar PR 2010. “An Introduction to material characterization”, Penram Int. publishing pvt. Ltd. ISBN978-81-87972-80-8.

22. Doug, Holly & Oleg, “SEM Microscope”. 10. M.T. Postek, K.S. Howard, A.H. Johnson and K.L. McMichael, Scanning Electron Microscopy: A Student’s Handbook, (Ladd Research Ind., Inc Williston, VT., 1980).

23. Watt IM, The Principles and Practice of Electron Microscopy, (Cambridge Univ. Press. Cambridge, England, 1985).

24. Lyman CE, Newbury DE, Goldstein JI, Williams DB, Romig AD, Armstrong JT, Echlin P, Fiori CE, Joy DC, Lifshin E, Peters KR, Scanning Electron Microscopy, X-Ray Microanalysis and Analytical Electron Microscopy: A Laboratory Workbook, (Plenum Press. New York, N.Y., 1990).

25. From Scanning Electron Microscopy and X-Ray Microanalysis, Joseph I. Goldstein, Plenum Press.

26. All the remaining figures are taken from google.com website.

27. Scanning Electron Microscopy: Physics of Image Formation and Microanalysis (Springer Series in Optical Sciences) by Ludwig Reimer and P.W. Hawkes (Hardcover - Oct 16, 1998)

28. A Guide to X-Ray Microanalysis, Oxford Microanalytical Instruments.

29. A Transmission Electron Microscope is anologous to a slide projector as indicated by Philips.

Received on 24.03.2021 Modified on 02.05.2021

Asian Journal of Pharmacy and Technology. 2021; 11(3):245-248.

DOI: 10.52711/2231-5713.2021.00040