Uses of Electron Microscope - Essay Example

 Uses of electron microscope Electron microscope is a type of microscope which uses a beam of electrons to create an image of the specimen. It is an essential component for scientific analysis of a variety of materials. Figure- 1: Electron Microscope (RSIC, 2008) Uses of electron microscope Examination under electron microscope yields following information of the specimen studied (CMRA, 2008) 1. Topography- The microscope enables observation of surface features of the specimen. The appearance, texture, hardness, reflectivity and many other aspects can be studied. 2. Morphology- The shape and size of the particles that make up the specimen can be observed through electron microscope. Also, direct relation between these structures and material properties like ductility, strength and reactivity can be assessed. 3. Composition- The microscope also helps to determine the elements and compounds that the object is composed of and the relative amounts of them. Also, direct relationship between composition and materials properties like melting point, reactivity and hardness can be determined. 4. Crystallographic information- Information as to how the atoms are arranged in the object and direct relation between these arrangements and materials properties like conductivity, electrical properties and strength can be derived (CMRA, 2008). History The light microscope was invented in the 17th century from the Galilean telescope. Antony van Leeuwenhoek, a Dutchman developed one of the early microscopes which consisted of a powerful convex lens and an adjustable holder for the object being studied. This instrument had a magnifying power of 400x and protozoa, spermatozoa, bacteria and shape of the red blood cells were discovered by Leeuwenhoek (FEI company, 2008). This microscope had only one lens and was called a single microscope. An improvement on this was compound microscope wherein another convex lens was added to magnify the image produced by the first lens. A modern light microscope has a magnification of as high as 1000x and thus enables resolution of objects separated by 0.0002mm (FEI Company, 2008). The resolving power of light microscope had 3 limiting factors: lenses, quality of lenses and the wavelength of light used for illumination. Some improvements in the light microscope were made using these aspects. Blue or ultraviolet light with shorter wavelength gave a small improvement. Further improvement in the resolution was noticed when the specimen and the front of the objective lens was immersed in a medium like oil with high refractive index (FEI Company, 2008). As early as middle of 19th century, microscopists realized that structures less than half a micrometre could not be resolved with a light microscope. At the same time researchers had hinted at the possibility of improvement in the resolution of microscope using electrons rather than light. This is because accelerated electrons behave in vacuum just like light, they travel in straight lines and have a wavelength which is about 100,000 times smaller than that of light. Also, electric and magnetic fields have the same effect on electrons as glass lenses and mirrors have on visible light (FEI Company, 2008). Two famous scientists of the 19th century merit attention as far as the beginning of electron microscopy is concerned. Hertz suggested that cathode rays were a form of wave motion and Weichert found that these rays could be concentrated into a small spot by the use of an axial magnetic field produced by a long solenoid. These suggestions came into light when in 1926; Busch demonstrated theoretically that a short solenoid converges a beam of electrons in the same way that glass can converge the light of the sun. He directly compared light and electron beams (CMRA, 2008). After this discovery, in 1931, the German engineers Ernst Ruska and Maximillion Knoll magnified images using electrons. However, the first prototype of electron microscope was built by Ruska in 1933. This model was capable of resolving to 50 nm. It had only 2 lens. Later, he added 3 lens and improved the resolution to 100nm. Ruska received Nobel Prize for Physics in 1986 for this invention (FEI Company, 2008). The first commercial electron microscope was manufactured by Metropolitan Vickers for Imperial College in London and it was called EM1. Disadvantages of this and other early models were that the electron beam had a very high current density which was concentrated into a very small area. The beam was very hot and charred the examined non-metallic specimens. However, researchers later found out that treating biological specimens with osmium and cutting very thin slices of the sample made electron microscopy viable. The first electron microscope was constructed by Eli Franklin Burton and his students in 1938 at the University of Toronto. This model of microscope was successful. The Second World War in 1939 hindered the progress in electron microscope. But 20 years later, newer models with resolution as high as 1nm were developed. Today, electron microscope has five magnetic lenses in the imaging system. It has a resolving power of 0.1 nm. Thus, magnifications of over 1 million times can be achieved (FEI Company, 2008). In an electron microscope, the electrons are accelerated in a vacuum until their wavelength is shortened. Shorter wavelengths can be produced by increasing the voltage. Beams of these fast-moving electrons are focused on an object. The object either absorbs or scatters these beams and forms an image on an electron-sensitive photographic plate (Merlos, Pennington, Lum, & Campbell, n.d.). Types of Electron Microscope There are basically 2 types of electron microscope: transmission type and the scanning type as discussed below. The Transmission Electron Microscope or TEM TEM is almost similar to light microscope but it uses electrons instead of light. There are four main parts to a TEM: an electron optical column, a vacuum system, the necessary electronics (lens supplies for focusing and deflecting the beam and the high voltage generator for the electron source), and software. This instrument can be compared to a slide projector. In the projector, light is emitted from a light source which is then passed through a condenser lens to convert it into a parallel beam which passes through the slide or rather object and is then focused as an enlarged image onto the screen by the objective lens. In case of electron microscope, a tungsten filament heated in vacuum produces electrons and replaces the light source of the projector (FEI Company, 2008). The magnetic lenses replace the glass lenses and the fluorescent screen replaces the projection screen. Electromagnetic lenses are variable. By varying the current through the lens coil, the focal length (which determines the magnification) can be varied. The fluorescent screen emits light when struck by electrons. The whole trajectory from source to screen is under vacuum. Otherwise, the electrons would collide with air molecules and be absorbed. The final image has to be viewed through a window in the projection chamber. One main disadvantage with TEM is that the specimen under study has to be very thin to allow the electrons to penetrate it. This is because an electron is nearly 2000x smaller and lighter than the smallest atom and hence electrons are easily stopped or deflected by matter. It is not possible to make all specimens thin enough to be studied under TEM. Specimens for the TEM are usually 0.5 micrometers or less thick. The higher the accelerating voltage in the gun, the higher the speed of electrons and consequently, the thicker the specimen that can be studied (FEI Company, 2008). The electron gun in TEM The electron gun comprises a filament made up of tungsten, a Wehnelt cylinder and an anode. All these three together are known as triode gun which is actually a stable source of electrons. The filament is hairpin shaped and heated to about 2700 degree centigrade. Electrons are extracted from the electron cloud round the filament by applying very high positive potential difference between the filament and the anode. The electron beam travels at several hundred thousand kilometres per second through a hole in the anode and emerges at the other side. The Wehnelt cylinder is at a different potential and bunches the electrons into a finely focused point (FEI Company, 2008). Figure- 2: Parts of TEM (Steve’s place, 2008) Specimen and electron bombardment in TEM Most of the specimens are not affected by electron bombardment as long as the bombardment is kept under control. Impingement of the electrons on the specimen can cause any one of the following (John Innes Centre, 2008): 1. Absorption: Some specimens absorb the electrons. This is based on the thickness and composition of the specimen which cause amplitude contrast in the image. 2. Scattering: Some electrons are scattered over small angles. The amount of scattering is dependent on the composition of the specimen. Scattering causes phase contrast in the image. 3. Diffraction: Diffraction of electrons occurs in crystalline specimens. The electrons are scattered in very distinct directions. 4. Reflection: Some of the electrons may be reflected by the specimen and such electrons are called 'backscattered electrons.' 5. Emission of electrons: The impinging electrons can cause the specimen itself to emit some electrons. Such electrons are known as secondary electrons. 6. Emission of X-rays: Impingement by electrons can lead to emission of X-rays by the specimen. The energy and wave length of the X- ray is related to the specimen's elemental composition. 7. Emission of photons or light: Some specimens emit photons after being impinged by electrons. This is known as cathodoluminescence. 8. Loss of energy: Some interaction between the electrons and the specimen can lead to loss of energy. This can be detected by Energy Loss Spectrometer. Absorption and scattering contribute to image formation in biological specimens while phase contrast and diffraction contrast lead to image formation in the crystalline specimens. The electromagnetic lenses in TEM Passing electrical current through coils creates electromagnetic field. Varying the current through the coils leads to magnification of the lens (Nobelprize.org, 2008). The electromagnetic lenses behave in the same way as glass lenses as far as aberrations are concerned. The 3 types of aberration are spherical aberration, chromatic aberration and astigmatism. In spherical aberration, the magnification in the centre of the lens differs from that at the edges. This type of aberration is largely determined by the lens design and manufacture. In chromatic aberration, the magnification of the lens varies with the wavelength of the electrons in the beam. The aberration can be reduced by keeping the accelerating voltage as stable as possible and using very thin specimens. In astigmatism, a circle in the specimen becomes an ellipse in the image. This can be corrected by using variable electromagnetic compensation coils (FEI company, 2008). Figure-3: Cross section of an electron microscope (Visual Merriam, 2008) The condenser lens system focuses only a necessary amount of electron beam onto the specimen. The objective lens produces an image of the specimen. This image is then magnified by the remaining imaging lenses and then projected onto the fluorescent screen. In case, the specimen is crystalline, a diffraction pattern will occur at a different point in the lens. This diffraction point is known as back focal plane. It is possible to enlarge the diffraction pattern and project this onto the fluorescent screen by varying the strength of the lens immediately below the objective lens (Nobelprize.org, 2008). Some TEMs like the Tecnai series, have four lenses: a diffraction lens, an intermediate lens and two projector lenses. Mordern TEMs have a water cooling system for their lenses to achieve the highest possible magnification (FEI company, 2008). Through the pathway from the filament to the fluorescent screen, the electron beam passes through a series of apertures with different diameters. Some electrons like the scattered electrons which are not useful for image formation are stopped. The diameter of the apertures in the condenser lens, the objective lens and the diffraction lens can be selected from outside the column as dictated by circumstances using a special holder carrying four different apertures (FEI company, 2008). The image in TEM The image falls on the fluorescent screen. This image can be observed through a large window in the projection chamber. A special fine grain focusing screen helps examine fine details of the image and assists correct focusing of the image. The focusing screen can be inserted into the beam and then observed through a high-quality 12x binocular viewer. Recording of the visualized image is done by replacing the fluorescent screen with a photographic film. Digital recording using a TV camera or video tape recorder is possible. This type of recording is useful for group viewing and instructional purposes (FEI company, 2008). Vacuum in TEM As previously discussed, electrons behave like light only when manipulated in vacuum. The whole column from gun to fluorescent screen and including the camera is evacuated to create vacuum. Various levels of vacuum are required: the highest level of vacuum is to be there around the specimen and the gun. This can be as high as the order of a ten millionth of a millimeter of mercury. The lowest level is found in the projection chamber and camera chamber. These levels are maintained by using different vacuum pumps. A number of airlocks and separation valves which are built in help maintain vacuum even when a specimen or photographic material is exchanged. The latest models in TEM have automated vacuum system and the vacuum level is continuously monitored and fully protected against faulty operation (FEI company, 2008). Maintenance of stable electronics in TEM It is important to maintain stability of the accelerating voltage and the current through the lenses. The power supply of a TEM is built in such a sophisticated way that output voltage or current does not deviate by more than one millionth of the value selected for a particular purpose. Control of electron microscope in TEM Powerful personal computers are employed to control, monitor and record the operating conditions of modern electron microscopes. These computers allow special techniques to be embedded in the instrument. The computer can also used for automatic back ups and results to be downloaded to other workstations. Updating of the microscope can be done easily by just upgrading the computer software (FEI company, 2008). Specimen preparation, orientation and manipulation in TEM TEM can be employed in any field of science and technology. The specimen should be small enough (almost as small as 3 mm) to be introduced into the evacuated microscope column and also thin enough to allow passage of electrons. These requirements can be achieved by proper preparation of the specimen. The technique used to prepare the specimen depends on the specimen, the type of analysis aimed at and also the type and model of microscope used (John Innes Centre, 2008). Some of the techniques used are described below: 1. Fixation: Most of the times fixation is done by gluteraldehyde and osmium tetroxide. The former cross links protein molecules, and the latter preserves lipids. Fixation prevents further deterioration of the specimen and makes the specimen appear as close to the living state. 2. Cryofixation: Rapid freezing of the specimen to temperatures less than liquid nitrogen temperatures makes ice out of water. This minimizes artefacts by preserving the specimen in a snapshot of its solution state. Infact, as an extension of this technology, cryo- electron microscopes have developed allowing any biological specimen to be studied in its native state. 3. Dehydration: This means removing water from the samples and replacing it by organic solvents like acetone or ethanol. Water can also be replaced with resin by infiltration. 4. Embedding: Infiltration of a tissue specimen with resin is known as embedding. The chemicals used for this purpose are araldite or LR White. Advantage of embedding is that after embedding, the specimen can be polymerized into a hardened block for subsequent sectioning. 5. Sectioning: Producing thin slices of the specimen is known as sectioning. For electron microscopy, it is important for the specimens to be as thin as 90nm so that they are semitransparent and allow electrons through them. Sectioning is done by using a glass or diamond knife and is done on an ultramicrotome. 6. Staining: This method of preparation is employed when a contrast is required between different structures. Heavy metals like lead and uranium are used for staining. They scatter the imaging electrons and thus give contrast. Biological specimens can be stained 'enbloc' before embedding or after sectioning as the requirement may be. 7. Freeze-fracture and freeze-etch: This is a special preparation method particularly useful for examining lipid membranes and the proteins encorporated in them. Firstly, the fresh tissue specimen or cell sample is frozen rapidly, i.e., cryofixed and then fractured by using a mirotome in the same frozen sate. After this, the temperature of the surface of the specimen is mildly increased to about -95 deg. centigrade for a few minutes allowing some ice to sublime and reveal microscopic details. This is known as 'etching'. This specimen can be further rotary-shadowed with evaporated platinum at low angle in a high vacuum evaporator. Stability of the replica coating is improved by a second coat of carbon, evaporated perpendicular to the average surface plane. The specimen is then returned to room temperature and pressure and then subjected to chemical digestion with acids, hypochlorite solution or SDS detergent to release the extremely fragile "shadowed" metal replica of the fracture surface. This floating replica is then thoroughly washed to remove residual chemicals. 8. Sputter coating: The sample is coated with an ultra-thin coating of electrically-conducting material like gold, gold/palladium, platinum and chromium by means of low vacuum coating of the sample to prevent charging of the specimen. Charging normally occurs due to electron irradiation and accumulation of static electric fields. Sputter coating also increases the amount of secondary electrons emitted from the sample surface and thus increases the signal to noise ratio. Scanning Transmission Electron Microscopy (STEM) Introduction of scanning facility in STEM allows to take advantage of the backscattered electrons as well as of the secondary electrons which are emitted during specimen bombardment. This facility in now a part of most of the TEMs manufactured. The lenses beneath the specimen greatly expand the number of possibilities for gathering information and hence including scanning facility in TEM is interesting. The Scanning Electron Microscope (SEM) Though it is not completely clear as to who invented the first SEM, the first published description was by Dr Max Knoll as early as 1935 (Purdue University, 2008). The SEM produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam (John Innes Centre, 2008). This is possible by scanning the electron beam across the sample in a 'raster' pattern. Detectors map the detected signals with beam position and build up the image (John Innes Centre, 2008). SEM consists of an electron optical column, a vacuum system and electronics similar to TEM. However, the column is considerably shorter because there are only three lenses to focus the electrons into a fine spot onto the specimen. Also, there are no lenses below the specimen. The specimen chamber is larger. This is because; the technique used in SEM does not impose any restriction on specimen size. The only restriction present is the size of the specimen chamber. The electronics unit, the lens supplies and the high voltage supplies are more compact than the TEM (Purdue University, 2008). Figure-4: Structure of TEM (Steve’s place, 2008) The electron gun in SEM This is similar to that of the TEM and consists of a tungsten filament and Wehnelt cylinder. The illumination system is also similar to the TEM and consists of electron gun, anode and condenser lenses (FEI Company, 2008). Specimen and electron bombardment in SEM When electrons strike the specimen, several phenomena occur. This has been discussed elaborately under TEM. Of these phenomena, five of them are used in ordinary SEM. These include, production of secondary electrons, reflection and backscattering of electrons, absorption of electrons, emission of X-rays and also emission of photons. It is important to note that all these phenomena depend on topography to some extent and also on the atomic number and the chemical state of the specimen (FEI Company, 2008). Electron detection in SEM The backscattered electrons and secondary electrons are detected by either a scintillation detector or a solid state detector. To be detected by a scintillation detector, the electrons strike a fluorescent screen which then emits light that is amplified and converted into an electrical signal by a photomultiplier tube. The solid state detector acts by amplifying the minute signal produced by the incoming electrons in a semiconductor device (FEI Company, 2008). Magnification and resolution in SEM The magnification is entirely determined by the electronic circuitry. This scans the beam over the specimen along with the beam over the fluorescent screen of the monitor. The magnification in SEM can be as high as 300,000x. Theoretically, resolution is determined by the beam diameter on the surface of the specimen. However, practically, the resolution depends on the properties of the specimen, the sample preparation technique used, beam intensity, scanning speed, distance from the last lens to the specimen, accelerating voltage and the angle of the specimen with respect to the detector (FEI Company, 2008). The image in SEM The image in SEM is completely electronically produced. There are 2 image monitors in SEM: one for observation by the operator, the other with an ordinary 35mm or polaroid photocamera. The images can be subject to all kinds of treatment like contrast enhancement, inversion, mixing of images from various detectors, color coding, image analysis and subtraction of the image from one detector from that produced by a different detector using modern electronics (FEI Company, 2008). Vacuum in SEM SEM requires a low vacuum which is produced either by an oil diffusion pump or a turbomolecular pump. These pumps are backed by a rotary pre-vacuum pump. There is not much of need for vacuum airlocks in SEM because the combination of pumps provides reasonable exchange times for specimen, filament and aperture (FEI Company, 2008). Electronics in SEM All the electronic units in SEM are housed in the microscope console. They are controlled by a personal computer. The voltages and currents required for the electron gun and condenser lens should be stable. Otherwise, the resolution power will be affected. Application of SEM SEM is commonly used in situations where information is required about the surface of a specimen. Specimen preparation, orientation and manipulation in SEM Any specimen that can withstand the vacuum of the chamber and the electron bombardment can be studied. Infact, many specimens can be presented for observation without any preparation. However, volatile components like water need to be removed prior to observation by drying methods. Non-conducting specimens need to be coated with a conducting layer to prevent charging up under electron bombardment. The most popular coating agent is gold, because the metal also gives a good yield of secondary electrons and hence a good quality image. Another advantage with gold coating is that, it gives a fine grain coating and is easily applied in a sputter coater. The quality of image in SEM is determined by the orientation and the distance of the specimen from the detectors and the final lens. These aspects can be controlled using the computer. .Differences between TEM and SEM 1. In TEM, the beam is static, in SEM it is not. 2. In TEM, the accelerating voltages are much lower when compared to SEM. This is because, penetration of the specimen is not necessary in TEM. The voltages are high in SEM. They range from 200 to 30,000 volts. 3. Preparation of specimens to be investigated by SEM is not as complicated as the preparation of specimens for TEM. TEM resolution is about an order of magnitude better than the SEM resolution. SEMs usually image conductive or semi-conductive materials best. Non- conductive materials can be imaged by coating the sample with a conductive layer of metal or by an environmental scanning electron microscope. For the TEM, samples are generally prepared by chemical exposure to give ultrastructural details, thus giving more scope for artefacts when compared to SEM. Environmental Scanning Electron Microscope (ESEM) Since it is difficult to visualize many samples like grease, adhesives, liquids, foods and gels, a new type of scanning electron microscope known as environmental scanning microscope was designed to study these substances easily without much preparation. Various biological cells, plants, asphalt, liquid suspensions and wood can be studied under this instrument without prior specimen preparation or gold coating (Merlos, Pennington, Lum, & Campbell, n.d.). These samples may also be observed in water vapor or other gasses such as CO2 or N2 at near atmospheric pressures. This is in contrast to scanning microscope in which samples have to be electrically conductive and not produce vapors in a vacuum. Disadvantages of electron microscope The most important disadvantage which is obvious is the cost of the instrument. Not only is the instrument costly to buy, it is also costly to maintain. This is because; electron microscope is very sensitive to vibration and magnetic fields and hence must be kept in buildings with special services. The instrument requires extremely stable high voltage supplies, extremely stable currents to each and every electromagnetic coil/lens, high/ultra-high vacuum systems which are being continuously pumped, and also a cooling water supply circulation through the lenses and pumps. Since the molecules that make up air scatter the electrons, the samples must be viewed in a vacuum. Viewing in vacuum necessitates elaborate preparation of the sample prior to viewing. Above all, significant training is necessary to operate and maintain the instrument. Artefacts in electron microscopy Artefacts are nothing but changes in the sample as a result of various steps involved in microscopy. Infact, every electron micrograph is an artefact by itself. Artefacts result due to changes in dimensions and molecular rearrangement. It is not possible to completely evade artefacts. However, their occurrence can be minimized by understanding the processes involved and by making informed choices of the best preparative procedures to use for each sample. Various presentations of artefacts include: loss of continuity in the membranes, distortion of organelles, disorganization of organelles, sharp bends or curves in filamentous structures like microtubules that are usually straight and empty spaces in the cytoplasm of cells. Experience helps microscopists to differentiate between artefacts and structure (FEI Company, 2008). References Merlos, M.M, Pennington, A., Lum, P., & Campbell, M. (n.d.). Electron microscopy. 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