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8, Scanning Electron Microscopy: Principle,, Components and Applications, Dr. M. Kannan, , Scanning Electron Microscope functions exactly as their optical counterparts, except that they use a focused beam of electrons instead of light to “image”, the specimen and gain information as to its structure and composition. Given, sufficient light, the unaided human eye can distinguish two points 0.2 mm apart., If the points are closer together, they will appear as a single point. This distance, is called the resolving power or resolution of the eye. Similarly, light microscopes, use visible light (400- 700nm) and transparent lenses to see objects as small as, about one micrometer (one millionth of a meter), such as a red blood cell (7 μm) or, a human hair (100 μm). Light microscope has a magnification of about 1000x and, enables the eye to resolve objects separated by 200 nm. Electron Microscopes were, developed due to the limitations of light microscopes, which are limited by the, physics of light. Electron Microscopes are capable of much higher magnifications, and have a greater resolving power than a light microscope, allowing it to see, much smaller objects at sub cellular, molecular and atomic level. The smallest the, wavelength of the illuminating sources is the best resolution of the microscope., De Broglie defined the wavelength of moving particles (electron) λ = h/mv, Where, λ= wavelength of particles, h= Planck,s constant, m= mass of the particle (electron),, v= velocity of the particles; after substituting the known values, λ = 12.3 Ao/V, The resolution of an optical microscope is defined as the shortest distance between, two points on a specimen that can still be distinguished by the observer or camera, system as separate entities. Resolution (r) = λ/ (2NA), Where λ is the imaging, wavelength, NA is objective numerical aperture., Magnification is the process of enlarging the appearance, not physical size, of, something. Magnification is defined as the ratio of image distance versus object, distance

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A Textbook on Fundamentals and Applications of Nanotechnology, , 82, , M= v/u, Where M= magnification, u= object distance, v= image distance, Magnification is also defined as the ratio of the resolving power of the eye to, resolving power (δ) of the microscope M= δ eye/ δ microscope, , Magnification, , Resolution, , Difference between light microscope and electron microscope, Sl., No., , Feature, , Light microscope, , Electron microscope, , 1., , Electromagnetic, spectrum, , Visible light, 400700nm, Colours visible, , Electrons, app. 4nm, Monochrome, , 2., , Maximum resolving, power, , app. 200nm, , 0.5nm with very fine, detail, , 3., , Maximum magnification, , x1000 to x1500, , x500000, , 4., , Radiation source, , Tungsten or quartz, halogen lamp, , High voltage (50kV), tungsten lamp,, Lanthanum hexaboride, , 5., , Lenses, , Glass, , Electro magnetics, , 6., , Interior, , Air-filled, , Vacuum, , 7., , Focusing screen, , Human eye (retina),, photographic film, , Fluorescent (TV) screen,, Photographic film, , 8., , Preparation of, specimens, , Temporary mounts, living or dead, , Tissues must be, dehydrated = dead, , 9., , Fixation, , Alcohol, , OsO4 or KMnO4, , 10., , Embedding medium, , Wax, , Resin, , 11., , Sectioning of specimen, , Hand or microtome, sectioning < 20µm slice, Whole cells visible, , Ultra microtome, sectioning, < 50nm slices, Parts of cells visible, , 12., , Staining, , Water soluble dyes, , Heavy metals, , 13., , Support for sample, , Glass slide, , Copper grid

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Scanning Electron Microscopy: Principle, Components and Applications, , 83, , Working principles of SEM, A beam of electrons is formed by the Electron Source and accelerated toward, the specimen using a positive electrical potential. The electron beam is confined, and focused using metal apertures and magnetic lenses into a thin, focused,, monochromatic beam. Electrons in the beam interact with the atoms of the, specimen, producing signals that contain information about its surface topography,, composition and other electrical properties. These interactions and effects are, detected and transformed into an image.

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84, , A Textbook on Fundamentals and Applications of Nanotechnology, , Components of SEM, Electron Column, The electron column is where the electron beam is generated under vacuum,, focused to a small diameter, and scanned across the surface of a specimen by, electromagnetic deflection coils. The lower portion of the column is called the, specimen chamber., Electron gun: An electron beam is thermionically emitted from an electron gun, fitted with a tungsten filament cathode. Tungsten has the highest melting point, and lowest vapour pressure of all metals, thereby allowing it to be heated for, electron emission, and because of its low cost. Other types of electron emitters, include lanthanum hexaboride (LaB6) cathodes, and field emission guns (FEG),, which may be of the cold-cathode type using tungsten single crystal emitters or, the thermally assisted Schottky type, using­emitters of zirconium oxide., Condenser Lenses: After the beam passes the anode it is influenced by two, condenser lenses that cause the beam to converge and pass through a focal point., In conjunction with the selected accelerating voltage the condenser lenses are, primarily responsible for determining the intensity of the electron beam when it, strikes the specimen., Apertures: The function of these apertures is to reduce and exclude extraneous, electrons in the lenses. The final lens aperture located below the scanning coils, determines the diameter or spot size of the beam at the specimen. The spot size on, the specimen will in part determine the resolution and depth of field. Decreasing, the spot size will allow for an increase in resolution and depth of field with a loss, of brightness., Scanning System: Images are formed by rastering the electron beam across the, specimen using deflection coils inside the objective lens. The stigmator or, astigmatism corrector is located in the objective lens and uses a magnetic field in, order to reduce aberrations of the electron beam. The electron beam should have a, circular cross section when it strikes the specimen however it is usually elliptical, thus the stigmator acts to control this problem., Specimen Chamber: The lower portion of the column is specimen stage and, controls are located. Specimens are mounted and secured onto the stage which, is controlled by a goniometer. The secondary electrons from the specimen are, attracted to the detector by a positive charge Manual stage controls are found on, the front side of the specimen chamber for x-y-z movement., Electron Detectors: Detectors collect the signal generated from interaction of beam, with specimen. Electronic detectors convert the signal into digital images and, most often collected signal are Secondary electrons by secondary electron detector, (Everhart–Thornley) Backscattered electrons by backscattered electrons detector, (Solid-State detector) and X-rays signal by Energy dispersive spectrometer (EDS), detector.

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Scanning Electron Microscopy: Principle, Components and Applications, , 85, , Vacuum System: Vacuum is produced by an oil diffusion pump backed by a, mechanical pump. In the diffusion pump a stream of hot oil vapor strikes and, pushes air molecules toward a mechanical pump that expels them from the, system. A mechanical pump and valve system are used to preevacuate the system, because a diffusion pump only operates after a vacuum is created. If the column is, in a gas filled environment, electrons will be scattered collide with air molecules, which would lead to reduction of the beam intensity and stability. Similarly, other, gas molecules, which could come from the sample or the microscope itself, could, form compounds and condense on the sample. This would lower the contrast, and obscure detail in the image. The chemical and thermal stability is necessary, for a well-functioning filament (gun pressure). The field emission gun, LaB6 and, tungsten filament requires ~ 10-10, ~ 10-6 and 10-4 Torr, respectively. Hence, gun, column of electron microscope require vacuum to facilitate the electrons signals, from the sample to the detector for better imaging.

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86, , A Textbook on Fundamentals and Applications of Nanotechnology, , How Scanning Electron Microscope (SEM) works, Ernst Ruska and Max Knoll developed first electron microscope during 1931with, resolution of 100nm and later by addition of electromagnetic lenses, brought the, resolution to 0.05nm. SEM is similar to the optical stereo-binocular microscope to, observe the morphology and shape of the specimen., ¾¾ The electron gun produces an electron beam when tungsten wire is heated, by current and accelerated by the anode., ¾¾ The beam travels in the vacuum column through electromagnetic fields, and lenses, which focus the beam down toward the sample., ¾¾ A mechanism of deflection coils enables to guide the beam so that it scans, the surface of the sample in a raster pattern., ¾¾ When the incident beam touches the surface of the sample and produces, signals viz.,, yy Secondary electrons (SE), yy Auger electrons, yy Back scattered electrons (BSE), yy Characteristic X – Rays, yy Cathodoluminescence, ¾¾ The emitted signals are trapped by electrical detectors, convert into digital, images and displayed on a screen as digital image., ¾¾ Provides information sample’s elemental composition, structural variation, and morphology., ¾¾ In the SEM, use much lower accelerating voltages to prevent beam, penetration into the sample since the requirement is generation of the, secondary electrons from the true surface structure of a sample. Therefore,, it is common to use low KV, in the range 1-5kV for biological samples, even, though the SEMs are capable of up to 30 kV.

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Scanning Electron Microscopy: Principle, Components and Applications, , 87, , Interaction of Electron Beam with Specimen:, When the primary electron beam interacts with the sample, the electrons lose, energy by repeated random scattering and absorption within a teardrop-shaped, volume of the specimen known as the interaction volume, which extends from less, than 100 nm to approximately 10 µm into the surface. The size of the interaction, volume depends on the electron’s landing energy, the atomic number of the, specimen and the specimen’s density. The energy exchange between the electron, beam and the sample results in the reflection of high-energy back scattered, electrons by elastic scattering, emission of low energy secondary, auger electrons, by inelastic scattering and the emission of electromagnetic radiation (X-rays and, cathodoluminescence), each of which can be detected by respective detectors. The, beam current absorbed by the specimen can also be detected and used to create, images of the distribution of specimen current. Electronic amplifiers of various, types are used to amplify the signals, electronic detectors convert the signals into, digital images and displayed on a computer monitor., , Backscattered electron: Those electrons, which are deflected, back in the direction, of the beam. The special detector in scanning and transmission electron

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88, , A Textbook on Fundamentals and Applications of Nanotechnology, , microscope traps these signals. These are used to discriminate areas of different, atomic numbered elements. Higher atomic numbered elements gives off more, backscattered electrons and appear brighter than lower numbered elements. It has, the resolution to the level of 1000 nm. These electrons have high energy., Secondary Electrons: These electrons are also collected with a special type of, detector used in SEM and TEM. They are used primarily to reveal topographical, feature of a specimen. It has the resolving power <10 nm. These electrons have, low energy., Auger Electrons: These are special types of low energy electrons that carry the, information about the chemical nature (atomic composition) of the specimen., These are generated from the upper layer of specimen. It is a powerful tool in, the material sciences for studying the distribution of the lighter numbered, atomic elements on the surface of the specimen. It has limited application in, biological sciences. It is specialized equipment known as scanning auger, electron spectrometer., Cathodoluminescent: This effect results when the energy of the impinging, electrons in converted into visible light. Certain types of compounds are capable, of cathode luminescence and detected by special types of detector. The resolution is, the similar to the light microscope., Bremsstrahlung: Two important types of x-ray may be generated when the beam, electron encounters the atoms of the specimen, continuous or bremsstrahlung, x-ray and characteristic x-ray are generated when incoming, beam passing, close to the atomic nucleus is slowed by the coulomb field of the nucleus with, the release of x- ray energy. The intensity of x-ray energy released depends, on how close the electron comes to the nucleus closer. The closer passes, decelerate the electron more and yield higher energy x-rays. These are used, to measure specimen mass thickness when quantitative analysis performed on, thin sections. These are continuous x-rays also known as background or white, radiation., Characteristic X-rays: When high energy beam electrons interact with the, shell electrons of the specimen atoms so that an inner shell electron is ejected., The removal of this electron temporarily ionizes the atom until an outer shell, electron drops into the vacancy to stabilize the atom. Since this electron comes, from a higher energy level, a certain amount of energy must be given off before, it will be accommodated in the inner shell. The energy is released as an x-ray,, the energy which equals the difference in energy between the two shells. Since, this x-ray is of a discrete energy level, rather than a continuous, this event, may be plotted as discrete peaks. Different elements will fill the vacancies, in shells in unique ways. This means that since each element will generate a, unique series of peaks, the spectrum may be used to identify the elements; such, discrete x-rays are termed characteristic x-rays. The equipment for detection, x-rays are energy dispersive x-ray (EDX) detector and Wavelength Dispersive, X-ray (WDX) Detector.

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89, , Scanning Electron Microscopy: Principle, Components and Applications, , Sample preparation for SEM, Step, , Chemical, , Temperature, , Time, , Repetitions, , Primary, fixation, , 2.5%, glutaraldehyde in, distilled water, , room or 0-4°C, , 2-4 hours or, microwave, , 1, , Wash, , distilled water, , room or 0-4°C, , 30 minutes, , 3-5, , Secondary, fixation, , 1-4% osmium, tetroxide in, distilled water, , room or 0-4°C, , 2-4 hours, , 1, , Wash, , distilled water, , room or 0-4°C, , 30 minutes, , 3-5, , Dehydration, , 25% ethanol, 50% ethanol, 70-75% ethanol, 90-95% ethanol, 100% ethanol, , room or 0-4°C, , 20 minutes, 20 minutes, 20 minutes, 20 minutes, 30 minutes, , 1, 1, 1, 1, 2, , Critical point dry, Mount on specimen stub with silver paste or graphite, Sputtering coat the biological sample with gold/palladium alloy for making them, conductive, Store stubs in desiccator and view the external surface with SEM, , SEM micrographs

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A Textbook on Fundamentals and Applications of Nanotechnology, , 90, , Applications of Scanning Electron Microscopy, Topography: The surface features of an object or “how it looks”, its texture; direct, relation between these features and materials properties (hardness, reflectivity..., etc.), Morphology: The shape and size of the particles making up the object; direct, relation between these structures and materials properties (ductility, strength,, reactivity...etc.), Composition: The elements and compounds that the object is composed of and the, relative amounts of them; direct relationship between composition and materials, properties (melting point, reactivity, hardness...etc.), Crystallographic Information: How the atoms are arranged in the object; direct, relation between these arrangements and materials properties (conductivity,, electrical properties, strength.etc.), , Advantages of SEM, ¾¾ It gives detailed 3D and topographical imaging and the versatile, information garnered from different detectors., ¾¾ This instrument works very fast., ¾¾ Modern SEMs allow for the generation of data in digital form., ¾¾ Most SEM samples require minimal preparation actions., , Disadvantages of SEM, ¾¾ SEMs are expensive and large., ¾¾ Special training is required to operate an SEM., ¾¾ The preparation of samples can result in artifacts., ¾¾ SEMs are limited to solid samples., ¾¾ SEMs carry a small risk of radiation exposure associated with the electrons, that scatter from beneath the sample surface., , References, Goldstein, J.I., Yakowitz, H.. Newbury, D.E Lifshin, E.. Colby, J.W Colby J.W. and. J.R., Coleman. 1975. Pratical Scanning Electron Microscopy: Electron and Ion, Microprobe Analysis., Loretto, M.H. 1984. Electron Beam Analysis of Materials, in Chapman and Hall,, London New York FEI. The Quanta 200 User’s Operation Manual 2nd, ed. (2004). I.M. Watt, The Principles and Practice of Electron Microscopy,, (Cambridge Univ. Press. Cambridge, England, 1985., Lyman, C.E., Newbury, D.E. Goldstein, J.I. Williams, D.B. Romig, A.D. Armstrong,, J.T. Echlin, P.. Fiori, C.E Joy, D.C. Lifshin E.and Klaus-Ruediger Peters,

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Scanning Electron Microscopy: Principle, Components and Applications, , 91, , 1 9 9 0 Scanning Electron Microscopy X-Ray Microanalysis and Analytical, Electron Microscopy: A Laboratory Workbook, Press. New York, N.Y., Postek, M.T.,. Howard, K.S Johnson A.H. McMichael K.L.1980. Scanning Electron, Microscopy: A Student’s Handbook, (Ladd Research Ind., Inc. Williston,, VT.)., , Questions, Fill in the blanks:, 1., , Electron microscope uses ................... as a source for making images., , 2., , Electron microscope was invented by ..................., , 3., , Resolution of unaided human eye is ..................., , 4., , Primary fixative used in sample preparation of SEM is ..................., , 5., , Formula for Resolution is ..................., , Choose the correct answer, 1., , What is the resolving power of light microscope?, ii) 200 �m, iv) 200nm, , 2., , v) 0.2 mm, , Which of the following is the first step in the processing of biological, material for transmission electron microscopy?, i), , Dehydration, , iii) Fixation, 3., , iii) 0.02m, , ii) Sectioning, iv) Embedding, , A vacuum is needed in the electron microscope to........................................., i), , Pull the electrons onto the ii) Eliminate molecules of nitrogen,, specimen, oxygen or carbon dioxide, , iii) Pull the specimen into the iv) Prevent, secondary, radiation, column, affecting the microscope control, panel, 4., , Which of the following statements about SEM is true?, i), , The specimen is, coated with gold, , iii) Quantum dots, 5., , usually ii) Carbon nanotubes, iv) All the above, , Ernst Ruska awarded Nobel prize during 1986 for their invention of______, i), , SEM, , ii) TEM, , iii) STM, , iv) AFM

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A Textbook on Fundamentals and Applications of Nanotechnology, , 92, , True or False, 1., , Secondary electrons are formed by collision of incident beam and sample, , 2., , In SEM copper grid is used as platform for sample analysis, , 3., , Electron microscope was invented in the year 1931 by Max Knoll and, Ernst Ruska., , 4., , In electron microscopy, the lenses used to magnify the image are made of, glasses, , 5., , 2.5% glutaraldehyde is used as primary fixative for SEM sample, preparation, , Short notes, 1., , What is meant by backscattered electron, , 2., , Light vs electron microscope differentiate, , 3., , Why vacuum is needed in electron microscope?, , 4., , Narrate the role of different components of SEM with illustration, , 5., , Advantage and disadvantage of SEM?, , Essay, 1., , Write in detail about essential components and working principle of, scanning electron microscope with diagram