{"id":303,"date":"2018-12-07T08:44:03","date_gmt":"2018-12-07T08:44:03","guid":{"rendered":"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/?post_type=chapter&#038;p=303"},"modified":"2018-12-07T09:08:26","modified_gmt":"2018-12-07T09:08:26","slug":"transmission-electron-microscop","status":"publish","type":"chapter","link":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/chapter\/transmission-electron-microscop\/","title":{"rendered":"Transmission electron microscop"},"content":{"raw":"<div><span style=\"float: right\"><a href=\"https:\/\/youtu.be\/PL3YdcGwOgE\" target=\"_blank\" rel=\"noopener\"><img src=\"http:\/\/epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/2018\/11\/download.png\" alt=\"epgp books\" width=\"75px\" height=\"75px;\" \/><\/a>\r\n<\/span><\/div>\r\n<div>\r\n\r\n&nbsp;\r\n\r\n&nbsp;\r\n\r\n&nbsp;\r\n\r\n&nbsp;\r\n\r\n<strong>Transmission electron microscopy (TEM):<\/strong>\r\n<ul>\r\n \t<li style=\"text-align: justify\">TEM is also known as conventional transmission electron microscopy or CTEM.<\/li>\r\n \t<li style=\"text-align: justify\">Max Knoll and Ernst Ruska invented in 1933 in Berlin.<\/li>\r\n \t<li style=\"text-align: justify\">Recent electron microscopy (based on transmission) commonly contains a beam column which is around 2.5m tall and has a 30cm diameter, and they its ability to attain a 2\u00c5 resolution.<\/li>\r\n \t<li style=\"text-align: justify\">This method is utilized for analysing<span style=\"text-align: initial;font-size: 1em\"> the surface structure i.e. morphology, surface imperfection i.e. defects, <\/span>crystal<span style=\"text-align: initial;font-size: 1em\"> structure of the atom, size of the particle and also samples composition.<\/span><\/li>\r\n<\/ul>\r\n<strong>\u00a0 \u00a0 Design of TEM similar to a light microscope-<\/strong>\r\n\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-307\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159.png\" alt=\"\" width=\"724\" height=\"404\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 1: Similarity of a transmission electron microscope with a wide field light microscope.<\/p>\r\n&nbsp;\r\n\r\n<strong>Working Principle-<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">An extremely thinnest sample is required for scanning in this method from which electron beam is passed through rendering its interaction with the sample as a result of which image is produced. Thus, this so formed image can be magnified and focused on the device used for an imaging, like a fluorescent screen, on a photographic film layer, or to be identified by a sensor like a CCD camera.<\/p>\r\n&nbsp;\r\n\r\n<strong>Instrumentation of TEM-<\/strong>\r\n<ul>\r\n \t<li>Source of electron<\/li>\r\n \t<li>Gun based on Thermionic Emission<\/li>\r\n \t<li>Beam of Electron<\/li>\r\n \t<li>Electromagnetic<span style=\"text-align: initial;font-size: 1em\"> lenses <\/span><\/li>\r\n \t<li><span style=\"text-align: initial;font-size: 1em\">Vacuum chamber<\/span><\/li>\r\n \t<li>Two Condensers lenses, objective and intermediate lens<\/li>\r\n \t<li>Sample holder and stage<\/li>\r\n \t<li>(Imaging Device) Phosphor or fluorescent screen<\/li>\r\n \t<li>Computer<\/li>\r\n<\/ul>\r\n<\/div>\r\n<div>\r\n\r\n<img class=\"aligncenter size-full wp-image-308\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160.png\" alt=\"\" width=\"230\" height=\"400\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 1: Instrumentation of TEM<\/p>\r\n&nbsp;\r\n\r\n<strong>Electron Gun:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Electrons can either be produced by thermionic emission or in a process called cold field emission. During thermionic emission, a very fine tip of a tungsten filament, a LaB6 crystal or a ZrO\/W Schottky emitter is heated by an electrical current flowing through the electron source enabling the escape of electrons. The electrons leaving the filament have a low energy and, therefore, need to be accelerated to the desired speed before entering the electron column. A high voltage between the electron source (cathode) and an anode plate is applied leading to an electrostatic field through which the electrons are guided and accelerated. During cold field emission, the electrons can escape from an extremely fine tungsten tip without heating (room temperature). The advantage of cold field emission sources is the very high yield of electrons and the very low chromatic aberration of the electrons allowing imaging at atomic resolution. These instruments are very costly and require particularly high vacuum.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The Electron gun working can be controlled based on 3 parameters:<\/span><\/p>\r\n\r\n<ul>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The accelerating voltage,<\/span><\/li>\r\n \t<li style=\"text-align: justify\">Current<span style=\"text-align: initial;font-size: 1em\"> of the filament (and therfore its temperature), <\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">And the Wehnelt cap bias voltage.<\/span><\/li>\r\n<\/ul>\r\n<\/div>\r\n<div>\r\n<p style=\"text-align: justify\">\u00a0 \u00a0The temperature of the filament tip is controlled by the filament current which in turn controls the amount of emitted electrons. The filament current is increased till the emitted electron no longer increases, which actually means that filament is saturated in order to maximize the emission.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">The passing current amongst the system of high voltage as well as earth is controlled by bias resistor setting which in turn is controlled by the gun bias. On small bias voltage when the wehnelt negative potential is compared to the filament is ineffective which means the electrons that are accelerated in anode direction is relatively with slight focusing. The beam is consequently spread and appears weak on the screen. When the biasing is increased then the focusing action is improved therefore the effective beam brightness is also increased; but, beyond a certain value the Wehnelt is so negative in comparison to the filament that the brightness starts to decrease because electrons are not permitted to emit from the filament or, in a case they are emitted so they are repelled back in the direction of the filament.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">The point on which the finest brightness of the beam is attained is determined by the distance amongst the Wehnelt and the filament.<\/p>\r\n&nbsp;\r\n\r\n<strong>Electromagnetic lenses:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Electromagnetic lenses consist of a huge bundle of windings of insulated copper wire, a soft iron cast and pole piece (figure 2A). A magnetic field is induced by the current in the winding and reaches its main strength at the pole piece of the lens. The accelerated electrons entering the magnetic field are deviated following the law of a charge passing a magnetic field. The direction of both magnetic field as well as electrons defines the resultant force which is always perpendicular to the plane. In conclusion, the electrons take a circular path through the lens system (figure 2B).<\/p>\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-309\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161.png\" alt=\"\" width=\"835\" height=\"298\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Fig 2: Electromagnetic lens. A) The magnetic field is strongest in the area of the pole piece; B) Electrons passing the magnetic field are deviated perpendicular to the plane defined by the magnetic field B and the velocity vector v.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Condenser lens system:<\/strong><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The beam diameter isreduced and controlled by the action of <\/span>condenser<span style=\"text-align: initial;font-size: 1em\"> lens system. The purpose of the first condenser C1 lens (or spot size) which is a strong lens is to de-magnify the electron source image by means of around X1\/100 to provide a small \u201cpoint\u201d source at the \u201ccrossover\u201d that is more coherent than the large (50 \u03bcm diameter) tip of the filament. The purpose of the second condenser C2 lens (brightness or intensity) which is a weaker lens is to project the de-magnified image of the source on top of the sample by a magnification of X2, giving an overall demagnification of X1\/50. Illumination <\/span>scatter<span style=\"text-align: initial;font-size: 1em\"> onto the screen is controlled by this lens. A part named condenser aperture is positioned just below or sometimes amongst the condenser lenses; its role is to collimate (i.e. making parallel) the beam of the electron as well as modification in its intensity.<\/span><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Objective and intermediate lenses:<\/strong><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The reason behind the back focal plane <\/span>being<span style=\"text-align: initial;font-size: 1em\"> very close to the lens itself is because the magnification factor of the objective lens is larger. <\/span>Aperture<span style=\"text-align: initial;font-size: 1em\"> of the objective (it\u2019s the middle aperture on the column) is mounted in the back-focal plane. Inside the first image plane beneath the sample selected area aperture is placed, that is underneath both the objective lens and the objective aperture.<\/span><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">By altering the first projector lens excitation (also known as <\/span>intermediate<span style=\"text-align: initial;font-size: 1em\"> lens or diffraction lens), either an image or a diffraction pattern is produced.<\/span><\/p>\r\n\r\n<\/div>\r\n<div>\r\n\r\n<img class=\"aligncenter size-full wp-image-310\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162.png\" alt=\"\" width=\"775\" height=\"519\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 3: Diagram showing either image or diffraction pattern formation.<\/p>\r\n&nbsp;\r\n\r\n<strong>Specimen holders and stages:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">In TEM, the electron column does not offer a lot of space for the sample. Additionally, the sample should be fine (thin) so that the electrons can penetrate the specimen and form an image. The average thickness of a biological specimen should be around 70 nm for a TEM with an acceleration voltage for the electrons of ~100 kV (the higher the voltage, the thicker specimens can be examined). Thin sections of the specimen are mounted on copper grids of 3 <\/span>mm<span style=\"text-align: initial;font-size: 1em\"> diameter, which are available in a wide variety of materials and mesh sizes. The grids with the sections on top are attached in a holder and introduced into the goniometer of the TEM through a vacuum <\/span>lock,<span style=\"text-align: initial;font-size: 1em\"> since the system always stays under high vacuum. The goniometer is the mechanical setup which enables highly precise and stable control of the specimen holder during imaging. Any drift or instability results in an un-sharp image, in particular at high magnifications (figure 4).<\/span><\/p>\r\n\r\n<\/div>\r\n<div>\r\n\r\n<img class=\"aligncenter size-full wp-image-311\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163.png\" alt=\"\" width=\"815\" height=\"457\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Fig 4: Thin sections of a specimen on a TEM grid, holder tip and complete specimen holder, which is introduced into the goniometer of the TEM through a vacuum lock.<\/p>\r\n&nbsp;\r\n\r\n<strong>Vacuum System:<\/strong>\r\n\r\n&nbsp;\r\n\r\nVacuum system is employed in electron microscopes for 4 reasons:\r\n<ul>\r\n \t<li style=\"text-align: justify\">As scattering of electrons is easy therefore electrons free mean path at atmospheric pressure is only around 1cm; however, at 10-6 Pa they can move around 6.5m.<\/li>\r\n \t<li style=\"text-align: justify\">The purpose of the vacuum system is to provide insulation amongst the filament of both anode and cathode as well as in the region around the field emitters, thus hampering undesirable electron gun electrical discharge.<\/li>\r\n \t<li style=\"text-align: justify\">In order to inhibit the oxidation and \u2018burning out\u2019 of the filament, oxygen is eliminated around the filament.<\/li>\r\n \t<li style=\"text-align: justify\">Samples contamination is decreased by reducing the interaction amongst beam of the electron and molecules of the gas.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<img class=\"aligncenter size-full wp-image-312\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164.png\" alt=\"\" width=\"763\" height=\"493\" \/>\r\n<div>\r\n<p style=\"text-align: justify\">Fig 5: Example of a vacuum system for a TEM. RP-rotary pump, TMP-turbo molecular pump, IGP-ion getter pump, ODF-oil diffusion pump.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">Various microscope parts are differently vacuumed as per their requirements. The gun requirement is 10-9 Pa vacuum, while the specimen requires 10-6 Pa and the projection chamber plus camera requires 10-5 Pa.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">Vacuums can be categorized as: rough (100 - 0.1 Pa), low (10<sup>-1<\/sup> - 10<sup>-4<\/sup> Pa), high (10<sup>-4<\/sup> - 10<sup>-7<\/sup> Pa), or ultrahigh (&lt; 10<sup>-7<\/sup> Pa).<\/p>\r\n&nbsp;\r\n\r\n<strong>Phosphor or fluorescent screen (Imaging Device):<\/strong>\r\n\r\n&nbsp;\r\n\r\nThere are 2 procedures for specimen observation in TEM as shown in fig.3.\r\n\r\n&nbsp;\r\n\r\n1.\u00a0 Image mode\r\n\r\n&nbsp;\r\n\r\n2.\u00a0 Diffraction mode\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">In case of image mode, the electron beam hitting the sample is controlled by condenser lens and aperture, the beam which is transmitted will be focused and enlarged by objective and projector lens thus the image is formed on the screen, with identifiable information in relation to the microstructure of the sample. In case of diffraction mode, at the fluorescent screen a diffraction pattern (of electron) is attained which has originated from the electron beam illuminated sample region. The pattern of diffraction is completely equal to that of a pattern of X\u2212ray diffraction. The spot pattern is produced by a single crystal on the screen whereas poly-crystal produces a pattern of powder or ring. The purpose of the image mode is to analyse microstructure, e.g. the grain size, and lattice defects, whereas the use of diffraction mode is to examine crystalline structure.<\/p>\r\n&nbsp;\r\n\r\n<strong>Image Modes of TEM-<\/strong>\r\n\r\n&nbsp;\r\n\r\nIn TEM, the 2 primary image modes vary in a style based on a technique an objective aperture is utilized as a filter in electron optics system are\r\n\r\n&nbsp;\r\n\r\n1. Bright field microscopy\r\n\r\n<span style=\"text-align: initial;font-size: 1em\">2. Dark field microscopy<\/span>\r\n\r\n<\/div>\r\n<div>\r\n\r\n<img class=\"aligncenter size-full wp-image-313\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165.png\" alt=\"\" width=\"711\" height=\"406\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 6: Two image modes of TEM<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">In bright field imaging, the image formed of a thin sample is by the electrons that permit through the film deprived of diffraction, the diaphragm is used to stop the diffracted electrons. In the corresponding dark field imaging mode, the image is formed by a diffracted beam. The technique is called as bright Field which is mainly sensitive to extended crystal lattice defects in an otherwise ordered crystal, such as dislocations. The electron rays corresponding to bright field and dark field imaging are shown in fig.6.<\/p>\r\n&nbsp;\r\n\r\n<strong>Electron interaction with matter-<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">The interaction between the electron beam and the sample is coulombic. The negatively charged electrons can interact strongly with the electron cloud in the solid and also the positively charged nucleus. In contrast x-rays are EM radiation and they only interact with the electron cloud. In TEM, for imaging purposes, only the forward scattered electrons are of interest. There are two main types of scattered radiation:<\/p>\r\n\r\n<ul>\r\n \t<li style=\"text-align: justify\">Elastic - this represents coherent scattering (mainly) with no loss of energy. There is also a phase relation with the incident radiation.<\/li>\r\n \t<li style=\"text-align: justify\">Inelastic - the energy of the scattered electrons is lower than the incident beam. These are also incoherent radiation with no phase relation with the incident radiation.<\/li>\r\n<\/ul>\r\n<strong>\u00a0 \u00a0 TEM sample preparation-<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Significant part of TEM is its sample preparation for the analysis. There are two main conditions for TEM sample preparation:<\/p>\r\n\r\n<ul>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron transparent sample must be used. If not the whole sample at least the ROI should be thin. The allowed thickness value for the metallic samples is 30 - 50 nm. Usually, 100 nm is an upper limit for the sample thickness.<\/span><\/li>\r\n \t<li style=\"text-align: justify\">The sample ought to be mechanically strong for treatment.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<p style=\"text-align: justify\">\u00a0 TEM samples are either self-supported or mounted on a grid for analysis. Copper grids are the most commonly used, though for high temperature work Mo grids are used. For nanoparticles and thin films a-C film is used as support. A-C has low contrast in the TEM and will not obscure the contrast arising from the specimen. Some typical TEM grids are shown in figure 7.<\/p>\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-314\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166.png\" alt=\"\" width=\"389\" height=\"375\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 7: Typical TEM grids<\/p>\r\n&nbsp;\r\n\r\n<strong>Thinning the sample by different techniques-<\/strong>\r\n\r\n&nbsp;\r\n\r\n<strong>Electrolytic polishing:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Electrolytic polishing is used for conducting samples like metals\/alloys in order to produce samples that are electron transparent. The initially sheet thickness can be around a few hundred \u03bcm. This can be prepared by rolling or grinding bulk specimens. Similarly, metal coatings on substrates can be peeled off and used for the final thinning. Thin discs can also be cut from bulk specimens. This process is called coring. These discs are thinned by electrolytic polishing. Electrolytic polishing technique is the window technique.<\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\">The sample is made the anode and a thin stainless sheet is made the cathode. The sample is immersed in the electrolyte, which is usually cooled by water or liquid nitrogen. Perchloric acid is usually used as the electrolyte. The sample edges are covered by lacquer to expose a 'window', hence the name. The experimental setup and the hole generation are shown in figure 8.<\/p>\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-316\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167.png\" alt=\"\" width=\"347\" height=\"325\" \/>\r\n<p style=\"text-align: center\"><span style=\"text-align: initial;font-size: 1em\">Fig 8: Window polishing technique.<\/span><\/p>\r\n\r\n<div>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">When a current is applied the material is dissolved from the anode (sample) and deposits on the cathode. The rate of dissolution depends on the current and applied voltage. The I - V characteristics are shown in figure 9. Depending on the current and voltage, there are three regimes - etching, polishing, and pitting. The edges are coated so that material removal will start within the window. Once a hole is formed within the window the sample is pulled out. The region around the hole is usually electron transparent and can be mounted on a TEM grid.<\/p>\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-317\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168.png\" alt=\"\" width=\"384\" height=\"233\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 9: I - V characteristics during polishing.<\/p>\r\n&nbsp;\r\n\r\n<strong>Ion milling technique:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">For non-conducting samples usually grinding and polishing steps are used in order to reduce sample thickness. Some an ultramicrotome is used in order to generate thin samples. These can be either electron transparent or can be used as the starting material for further thinning. The schematic of the technique is shown in Fig 10. For samples, where ultramicrotome cannot be used then a standard tripod polisher is used in order to thin the sample. This produces samples that are a few nm thick. The final polishing step is done by an ion beam miller.<\/p>\r\n\r\n<\/div>\r\n<img class=\"aligncenter size-full wp-image-318\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169.png\" alt=\"\" width=\"289\" height=\"461\" \/>\r\n<div>\r\n<p style=\"text-align: center\">Fig 10: Schematic of ultramicrotome technique.<\/p>\r\n&nbsp;\r\n\r\n<img class=\"aligncenter size-full wp-image-319\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170.png\" alt=\"\" width=\"327\" height=\"419\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">fig 11: The schematic of the ion beam miller and an actual instrument<\/p>\r\n\r\n<\/div>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The sample is bombarded by high energy ions or neutral atoms. <\/span>Usually<span style=\"text-align: initial;font-size: 1em\"> Ar ions are used and they are formed by passing the Ar gas <\/span>though<span style=\"text-align: initial;font-size: 1em\"> a high voltage (4 - 6 keV ). The sample is held in <\/span>vacuum<span style=\"text-align: initial;font-size: 1em\"> and also usually cooled by liquid nitrogen. The ions are incident on the sample and sputter material away. To minimize ion penetration the beam is usually incident at a low angle (\u2248 20\u00b0) though if the angle is very small the sputter rate is small. <\/span>Ion<span style=\"text-align: initial;font-size: 1em\"> beam is highly controlled and a localized process but it is <\/span>time consuming<span style=\"text-align: initial;font-size: 1em\">. Sputter rates are usually a few A\u00b0 per second so that creating an electron transparent sample can take hours, especially if the initial thickness is high.<\/span><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Cross section sample preparation:<\/strong><\/p>\r\n&nbsp;\r\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Slices from the sample are cut using a diamond slicer. These slices are placed between spacer layers and then glued on to a grid. The slices are glued in such a way that the interface is parallel to the slot in the grid. This sample is then thinned by standard tripod polishing until it is a few \u03bcm thick. The final sample is thinned using <\/span>a ion<span style=\"text-align: initial;font-size: 1em\"> beam miller to create an electron transparent sample.<\/span><\/p>\r\n\r\n<div>\r\n\r\n<img class=\"aligncenter size-full wp-image-320\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171.png\" alt=\"\" width=\"365\" height=\"266\" \/>\r\n\r\n&nbsp;\r\n<p style=\"text-align: center\">Fig 12: Cross sectional sample preparation.<\/p>\r\n&nbsp;\r\n\r\n<strong>Replica technique:<\/strong>\r\n\r\n&nbsp;\r\n<p style=\"text-align: justify\">Replica technique is used for studying bulk specimens which cannot be destroyed to prepare electron specimens. It is also useful for studying surface topography features and precipitates though SEM techniques have gradually replaced replica sample preparation. A replica of the sample surface is prepared using a plastic mold. The mold is then removed from the surface and the surface of the specimen is replicated by the surface of the plastic. A thin film of carbon or metal like Cr, Pt is evaporated on the surface of the plastic. Sometimes the evaporation is done from an oblique angle, shadow evaporation, to enhance the contrast. The plastic is removed by dissolving in a suitable solvent and the film is then floated on to a grid for analysis.<\/p>\r\n\r\n<\/div>\r\n<img class=\"aligncenter size-full wp-image-321\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172.png\" alt=\"\" width=\"533\" height=\"318\" \/>\r\n<div>\r\n<p style=\"text-align: center\">Fig 13: Replica technique for sample preparation.<\/p>\r\n\r\n<\/div>\r\n<table>\r\n<tbody>\r\n<tr>\r\n<td><strong>you can view video on Transmission Electron Microscopy<\/strong><\/td>\r\n<td><a href=\"https:\/\/youtu.be\/PL3YdcGwOgE\" target=\"_blank\" rel=\"noopener\"><img class=\"alignnone wp-image-120\" src=\"http:\/\/epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/2018\/11\/download.png\" alt=\"\" width=\"36\" height=\"36\" \/><\/a><\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n<strong style=\"text-align: initial;font-size: 1em\">References<\/strong>\r\n<ol>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">coen.boisestate.edu\/faculty-staff\/files\/2012\/01\/TEM.pdf<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Physical Principles of Electron Microscopy, Ray F. Egerton, Springer Verlag, 2007.<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Griffith G. (1993). Fine Structure Immunocytochemistry. New York, Berlin, Heidelberg. Springer Verlag. ISBN0-387-54805-X.<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron microscopy methods and <\/span>protocols \/ ed<span style=\"text-align: initial;font-size: 1em\">. by M.A. Nasser Hajibagheri. - Totowa, N.J. : Humana Press, cop. 1999. (Methods in molecular <\/span>biology ;<span style=\"text-align: initial;font-size: 1em\"> vol. 117)<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron <\/span>microscopy :<span style=\"text-align: initial;font-size: 1em\"> methods and protocols. - 2nd ed. \/ ed. by John Kuo - Totowa, N.J. : Humana Press, 2007. (Methods in molecular <\/span>biology ;<span style=\"text-align: initial;font-size: 1em\"> 369)<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron microscopy : principles and techniques for biologists \/ John J. Bozzola, Lonnie D.\u00a0<\/span><span style=\"text-align: initial;font-size: 1em\">Russell. - <\/span>Boston :<span style=\"text-align: initial;font-size: 1em\"> Jones and Bartlett, 1991. (The Jones and Bartlett series in biology)<\/span><\/li>\r\n \t<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Introduction to electron microscopy, Andres Kaech, April 2013.<\/span><\/li>\r\n<\/ol>\r\n<strong>\u00a0 \u00a0 Review your learning<\/strong>\r\n\r\n&nbsp;\r\n\r\n1)\u00a0\u00a0\u00a0\u00a0\u00a0 Why are thin sections of specimens necessary in TEM?\r\n\r\n&nbsp;\r\n\r\na)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons are negatively charged\r\n\r\nb)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have a wave nature\r\n\r\nc)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have no mass\r\n\r\nd)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have a poor penetrating power.\r\n\r\n&nbsp;\r\n\r\n2)\u00a0\u00a0\u00a0\u00a0\u00a0 Why TEM images have much higher resolution than images from light microscope?\r\n\r\n&nbsp;\r\n\r\na)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 TEM is much greater in size than light microscope\r\n\r\nb)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons travelling as waves have wavelength much shorter than visible light\r\n\r\nc)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 TEM can achieve greater magnification\r\n\r\nd)\u00a0\u00a0\u00a0\u00a0\u00a0 The fluorescent screen of TEM can generate high resolution images\r\n\r\n&nbsp;\r\n\r\n3)\u00a0\u00a0\u00a0\u00a0\u00a0 Which of the following is the correct pathway of electrons in the TEM?\r\n\r\n&nbsp;\r\n\r\na)\u00a0 \u00a0 \u00a0 Anode \u2192 electromagnetic lens system \u2192 sample \u2192 fluorescent screen\r\n\r\nb)\u00a0\u00a0\u00a0\u00a0\u00a0 Anode \u2192 electromagnetic lens system \u2192 sample \u2192 electromagnetic lens system \u2192 fluorescent screen\r\n\r\nc)\u00a0 \u00a0 \u00a0 Cathode \u2192 electromagnetic lens system \u2192 sample \u2192 electromagnetic lens system \u2192 fluorescent screen\r\n\r\nd)\u00a0\u00a0\u00a0\u00a0\u00a0 Cathode \u2192 electromagnetic lens system \u2192 sample \u2192 fluorescent screen\r\n\r\n&nbsp;\r\n\r\n4) What should be done right after the TEM column is shown to be evacuated?\r\n\r\n&nbsp;\r\n\r\na) Insert the sample holder\r\n\r\nb) Further insert the sample holder\r\n\r\nc) Remove the dummy holder\r\n\r\nd) Shift the beam\r\n\r\n&nbsp;\r\n\r\n5) Before loading the sample, the following softwares have to be turned on, except\u2026\r\n\r\n&nbsp;\r\n\r\na) Electron gun tilt\/shift\r\n\r\nb) TEM imaging and analysis\r\n\r\nc) Microscope user interface\r\n\r\nd) Digital Micrograph\r\n\r\n&nbsp;\r\n\r\n<strong>True\/False:<\/strong>\r\n\r\n&nbsp;\r\n\r\n1) Ultra-violet light is applied to the sample to warm up the sample.\r\n\r\n<span style=\"font-size: 1em\">2) The main purpose of cutting extremely thin slices of samples is for better observation of intracellular components instead of extracellular components.<\/span>\r\n\r\n<span style=\"font-size: 1em\">3) TEM cannot be used to examine <\/span>live<span style=\"font-size: 1em\"> specimen.<\/span>\r\n\r\n<span style=\"font-size: 1em\">4) We should turn off the light before <\/span>examination<span style=\"font-size: 1em\"> of <\/span>sample<span style=\"font-size: 1em\"> using the fluorescent screen.<\/span>\r\n\r\n<span style=\"font-size: 1em\">5) We need to do the alignment of the electron gun, beam and rotation center every time we use <\/span>the\u00a0<span style=\"font-size: 1em\">TEM<\/span>\r\n\r\n&nbsp;\r\n\r\n<strong>Fill in the Blanks:<\/strong>\r\n\r\n&nbsp;\r\n\r\n1)\u00a0\u00a0 The gun requires the vacuum of the order of\u00a0\u00a0 \u2026\u2026\u2026\u2026\u2026.\r\n\r\n2) The specimen requires the vacuum of the order of \u2026\u2026\u2026\u2026..\r\n\r\n<span style=\"font-size: 1em\">3) Projection chamber and camera requires the vacuum of the order of \u2026\u2026<\/span>\r\n\r\n&nbsp;\r\n\r\n<strong>Long type questions:<\/strong>\r\n\r\n&nbsp;\r\n\r\n1) Explain the working principle of TEM\r\n\r\n<span style=\"font-size: 1em\">2) Describe various types of pumps used to achieve vacuum?<\/span>","rendered":"<div><span style=\"float: right\"><a href=\"https:\/\/youtu.be\/PL3YdcGwOgE\" target=\"_blank\" rel=\"noopener\"><img decoding=\"async\" src=\"http:\/\/epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/2018\/11\/download.png\" alt=\"epgp books\" width=\"75px\" height=\"75px;\" \/><\/a><br \/>\n<\/span><\/div>\n<div>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Transmission electron microscopy (TEM):<\/strong><\/p>\n<ul>\n<li style=\"text-align: justify\">TEM is also known as conventional transmission electron microscopy or CTEM.<\/li>\n<li style=\"text-align: justify\">Max Knoll and Ernst Ruska invented in 1933 in Berlin.<\/li>\n<li style=\"text-align: justify\">Recent electron microscopy (based on transmission) commonly contains a beam column which is around 2.5m tall and has a 30cm diameter, and they its ability to attain a 2\u00c5 resolution.<\/li>\n<li style=\"text-align: justify\">This method is utilized for analysing<span style=\"text-align: initial;font-size: 1em\"> the surface structure i.e. morphology, surface imperfection i.e. defects, <\/span>crystal<span style=\"text-align: initial;font-size: 1em\"> structure of the atom, size of the particle and also samples composition.<\/span><\/li>\n<\/ul>\n<p><strong>\u00a0 \u00a0 Design of TEM similar to a light microscope-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-307\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159.png\" alt=\"\" width=\"724\" height=\"404\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159.png 724w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159-300x167.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159-65x36.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159-225x126.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-159-350x195.png 350w\" sizes=\"auto, (max-width: 724px) 100vw, 724px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 1: Similarity of a transmission electron microscope with a wide field light microscope.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Working Principle-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">An extremely thinnest sample is required for scanning in this method from which electron beam is passed through rendering its interaction with the sample as a result of which image is produced. Thus, this so formed image can be magnified and focused on the device used for an imaging, like a fluorescent screen, on a photographic film layer, or to be identified by a sensor like a CCD camera.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Instrumentation of TEM-<\/strong><\/p>\n<ul>\n<li>Source of electron<\/li>\n<li>Gun based on Thermionic Emission<\/li>\n<li>Beam of Electron<\/li>\n<li>Electromagnetic<span style=\"text-align: initial;font-size: 1em\"> lenses <\/span><\/li>\n<li><span style=\"text-align: initial;font-size: 1em\">Vacuum chamber<\/span><\/li>\n<li>Two Condensers lenses, objective and intermediate lens<\/li>\n<li>Sample holder and stage<\/li>\n<li>(Imaging Device) Phosphor or fluorescent screen<\/li>\n<li>Computer<\/li>\n<\/ul>\n<\/div>\n<div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-308\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160.png\" alt=\"\" width=\"230\" height=\"400\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160.png 230w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160-173x300.png 173w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160-65x113.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-160-225x391.png 225w\" sizes=\"auto, (max-width: 230px) 100vw, 230px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 1: Instrumentation of TEM<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Electron Gun:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Electrons can either be produced by thermionic emission or in a process called cold field emission. During thermionic emission, a very fine tip of a tungsten filament, a LaB6 crystal or a ZrO\/W Schottky emitter is heated by an electrical current flowing through the electron source enabling the escape of electrons. The electrons leaving the filament have a low energy and, therefore, need to be accelerated to the desired speed before entering the electron column. A high voltage between the electron source (cathode) and an anode plate is applied leading to an electrostatic field through which the electrons are guided and accelerated. During cold field emission, the electrons can escape from an extremely fine tungsten tip without heating (room temperature). The advantage of cold field emission sources is the very high yield of electrons and the very low chromatic aberration of the electrons allowing imaging at atomic resolution. These instruments are very costly and require particularly high vacuum.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The Electron gun working can be controlled based on 3 parameters:<\/span><\/p>\n<ul>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The accelerating voltage,<\/span><\/li>\n<li style=\"text-align: justify\">Current<span style=\"text-align: initial;font-size: 1em\"> of the filament (and therfore its temperature), <\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">And the Wehnelt cap bias voltage.<\/span><\/li>\n<\/ul>\n<\/div>\n<div>\n<p style=\"text-align: justify\">\u00a0 \u00a0The temperature of the filament tip is controlled by the filament current which in turn controls the amount of emitted electrons. The filament current is increased till the emitted electron no longer increases, which actually means that filament is saturated in order to maximize the emission.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">The passing current amongst the system of high voltage as well as earth is controlled by bias resistor setting which in turn is controlled by the gun bias. On small bias voltage when the wehnelt negative potential is compared to the filament is ineffective which means the electrons that are accelerated in anode direction is relatively with slight focusing. The beam is consequently spread and appears weak on the screen. When the biasing is increased then the focusing action is improved therefore the effective beam brightness is also increased; but, beyond a certain value the Wehnelt is so negative in comparison to the filament that the brightness starts to decrease because electrons are not permitted to emit from the filament or, in a case they are emitted so they are repelled back in the direction of the filament.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">The point on which the finest brightness of the beam is attained is determined by the distance amongst the Wehnelt and the filament.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Electromagnetic lenses:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Electromagnetic lenses consist of a huge bundle of windings of insulated copper wire, a soft iron cast and pole piece (figure 2A). A magnetic field is induced by the current in the winding and reaches its main strength at the pole piece of the lens. The accelerated electrons entering the magnetic field are deviated following the law of a charge passing a magnetic field. The direction of both magnetic field as well as electrons defines the resultant force which is always perpendicular to the plane. In conclusion, the electrons take a circular path through the lens system (figure 2B).<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-309\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161.png\" alt=\"\" width=\"835\" height=\"298\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161.png 835w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161-300x107.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161-768x274.png 768w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161-65x23.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161-225x80.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-161-350x125.png 350w\" sizes=\"auto, (max-width: 835px) 100vw, 835px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Fig 2: Electromagnetic lens. A) The magnetic field is strongest in the area of the pole piece; B) Electrons passing the magnetic field are deviated perpendicular to the plane defined by the magnetic field B and the velocity vector v.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Condenser lens system:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The beam diameter isreduced and controlled by the action of <\/span>condenser<span style=\"text-align: initial;font-size: 1em\"> lens system. The purpose of the first condenser C1 lens (or spot size) which is a strong lens is to de-magnify the electron source image by means of around X1\/100 to provide a small \u201cpoint\u201d source at the \u201ccrossover\u201d that is more coherent than the large (50 \u03bcm diameter) tip of the filament. The purpose of the second condenser C2 lens (brightness or intensity) which is a weaker lens is to project the de-magnified image of the source on top of the sample by a magnification of X2, giving an overall demagnification of X1\/50. Illumination <\/span>scatter<span style=\"text-align: initial;font-size: 1em\"> onto the screen is controlled by this lens. A part named condenser aperture is positioned just below or sometimes amongst the condenser lenses; its role is to collimate (i.e. making parallel) the beam of the electron as well as modification in its intensity.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Objective and intermediate lenses:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The reason behind the back focal plane <\/span>being<span style=\"text-align: initial;font-size: 1em\"> very close to the lens itself is because the magnification factor of the objective lens is larger. <\/span>Aperture<span style=\"text-align: initial;font-size: 1em\"> of the objective (it\u2019s the middle aperture on the column) is mounted in the back-focal plane. Inside the first image plane beneath the sample selected area aperture is placed, that is underneath both the objective lens and the objective aperture.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">By altering the first projector lens excitation (also known as <\/span>intermediate<span style=\"text-align: initial;font-size: 1em\"> lens or diffraction lens), either an image or a diffraction pattern is produced.<\/span><\/p>\n<\/div>\n<div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-310\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162.png\" alt=\"\" width=\"775\" height=\"519\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162.png 775w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162-300x201.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162-768x514.png 768w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162-65x44.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162-225x151.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-162-350x234.png 350w\" sizes=\"auto, (max-width: 775px) 100vw, 775px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 3: Diagram showing either image or diffraction pattern formation.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Specimen holders and stages:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">In TEM, the electron column does not offer a lot of space for the sample. Additionally, the sample should be fine (thin) so that the electrons can penetrate the specimen and form an image. The average thickness of a biological specimen should be around 70 nm for a TEM with an acceleration voltage for the electrons of ~100 kV (the higher the voltage, the thicker specimens can be examined). Thin sections of the specimen are mounted on copper grids of 3 <\/span>mm<span style=\"text-align: initial;font-size: 1em\"> diameter, which are available in a wide variety of materials and mesh sizes. The grids with the sections on top are attached in a holder and introduced into the goniometer of the TEM through a vacuum <\/span>lock,<span style=\"text-align: initial;font-size: 1em\"> since the system always stays under high vacuum. The goniometer is the mechanical setup which enables highly precise and stable control of the specimen holder during imaging. Any drift or instability results in an un-sharp image, in particular at high magnifications (figure 4).<\/span><\/p>\n<\/div>\n<div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-311\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163.png\" alt=\"\" width=\"815\" height=\"457\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163.png 815w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163-300x168.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163-768x431.png 768w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163-65x36.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163-225x126.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-163-350x196.png 350w\" sizes=\"auto, (max-width: 815px) 100vw, 815px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Fig 4: Thin sections of a specimen on a TEM grid, holder tip and complete specimen holder, which is introduced into the goniometer of the TEM through a vacuum lock.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Vacuum System:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>Vacuum system is employed in electron microscopes for 4 reasons:<\/p>\n<ul>\n<li style=\"text-align: justify\">As scattering of electrons is easy therefore electrons free mean path at atmospheric pressure is only around 1cm; however, at 10-6 Pa they can move around 6.5m.<\/li>\n<li style=\"text-align: justify\">The purpose of the vacuum system is to provide insulation amongst the filament of both anode and cathode as well as in the region around the field emitters, thus hampering undesirable electron gun electrical discharge.<\/li>\n<li style=\"text-align: justify\">In order to inhibit the oxidation and \u2018burning out\u2019 of the filament, oxygen is eliminated around the filament.<\/li>\n<li style=\"text-align: justify\">Samples contamination is decreased by reducing the interaction amongst beam of the electron and molecules of the gas.<\/li>\n<\/ul>\n<\/div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-312\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164.png\" alt=\"\" width=\"763\" height=\"493\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164.png 763w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164-300x194.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164-65x42.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164-225x145.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-164-350x226.png 350w\" sizes=\"auto, (max-width: 763px) 100vw, 763px\" \/><\/p>\n<div>\n<p style=\"text-align: justify\">Fig 5: Example of a vacuum system for a TEM. RP-rotary pump, TMP-turbo molecular pump, IGP-ion getter pump, ODF-oil diffusion pump.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Various microscope parts are differently vacuumed as per their requirements. The gun requirement is 10-9 Pa vacuum, while the specimen requires 10-6 Pa and the projection chamber plus camera requires 10-5 Pa.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Vacuums can be categorized as: rough (100 &#8211; 0.1 Pa), low (10<sup>-1<\/sup> &#8211; 10<sup>-4<\/sup> Pa), high (10<sup>-4<\/sup> &#8211; 10<sup>-7<\/sup> Pa), or ultrahigh (&lt; 10<sup>-7<\/sup> Pa).<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Phosphor or fluorescent screen (Imaging Device):<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>There are 2 procedures for specimen observation in TEM as shown in fig.3.<\/p>\n<p>&nbsp;<\/p>\n<p>1.\u00a0 Image mode<\/p>\n<p>&nbsp;<\/p>\n<p>2.\u00a0 Diffraction mode<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">In case of image mode, the electron beam hitting the sample is controlled by condenser lens and aperture, the beam which is transmitted will be focused and enlarged by objective and projector lens thus the image is formed on the screen, with identifiable information in relation to the microstructure of the sample. In case of diffraction mode, at the fluorescent screen a diffraction pattern (of electron) is attained which has originated from the electron beam illuminated sample region. The pattern of diffraction is completely equal to that of a pattern of X\u2212ray diffraction. The spot pattern is produced by a single crystal on the screen whereas poly-crystal produces a pattern of powder or ring. The purpose of the image mode is to analyse microstructure, e.g. the grain size, and lattice defects, whereas the use of diffraction mode is to examine crystalline structure.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Image Modes of TEM-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>In TEM, the 2 primary image modes vary in a style based on a technique an objective aperture is utilized as a filter in electron optics system are<\/p>\n<p>&nbsp;<\/p>\n<p>1. Bright field microscopy<\/p>\n<p><span style=\"text-align: initial;font-size: 1em\">2. Dark field microscopy<\/span><\/p>\n<\/div>\n<div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-313\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165.png\" alt=\"\" width=\"711\" height=\"406\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165.png 711w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165-300x171.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165-65x37.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165-225x128.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-165-350x200.png 350w\" sizes=\"auto, (max-width: 711px) 100vw, 711px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 6: Two image modes of TEM<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">In bright field imaging, the image formed of a thin sample is by the electrons that permit through the film deprived of diffraction, the diaphragm is used to stop the diffracted electrons. In the corresponding dark field imaging mode, the image is formed by a diffracted beam. The technique is called as bright Field which is mainly sensitive to extended crystal lattice defects in an otherwise ordered crystal, such as dislocations. The electron rays corresponding to bright field and dark field imaging are shown in fig.6.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Electron interaction with matter-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">The interaction between the electron beam and the sample is coulombic. The negatively charged electrons can interact strongly with the electron cloud in the solid and also the positively charged nucleus. In contrast x-rays are EM radiation and they only interact with the electron cloud. In TEM, for imaging purposes, only the forward scattered electrons are of interest. There are two main types of scattered radiation:<\/p>\n<ul>\n<li style=\"text-align: justify\">Elastic &#8211; this represents coherent scattering (mainly) with no loss of energy. There is also a phase relation with the incident radiation.<\/li>\n<li style=\"text-align: justify\">Inelastic &#8211; the energy of the scattered electrons is lower than the incident beam. These are also incoherent radiation with no phase relation with the incident radiation.<\/li>\n<\/ul>\n<p><strong>\u00a0 \u00a0 TEM sample preparation-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Significant part of TEM is its sample preparation for the analysis. There are two main conditions for TEM sample preparation:<\/p>\n<ul>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron transparent sample must be used. If not the whole sample at least the ROI should be thin. The allowed thickness value for the metallic samples is 30 &#8211; 50 nm. Usually, 100 nm is an upper limit for the sample thickness.<\/span><\/li>\n<li style=\"text-align: justify\">The sample ought to be mechanically strong for treatment.<\/li>\n<\/ul>\n<\/div>\n<p style=\"text-align: justify\">\u00a0 TEM samples are either self-supported or mounted on a grid for analysis. Copper grids are the most commonly used, though for high temperature work Mo grids are used. For nanoparticles and thin films a-C film is used as support. A-C has low contrast in the TEM and will not obscure the contrast arising from the specimen. Some typical TEM grids are shown in figure 7.<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-314\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166.png\" alt=\"\" width=\"389\" height=\"375\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166.png 389w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166-300x289.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166-65x63.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166-225x217.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-166-350x337.png 350w\" sizes=\"auto, (max-width: 389px) 100vw, 389px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 7: Typical TEM grids<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Thinning the sample by different techniques-<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><strong>Electrolytic polishing:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Electrolytic polishing is used for conducting samples like metals\/alloys in order to produce samples that are electron transparent. The initially sheet thickness can be around a few hundred \u03bcm. This can be prepared by rolling or grinding bulk specimens. Similarly, metal coatings on substrates can be peeled off and used for the final thinning. Thin discs can also be cut from bulk specimens. This process is called coring. These discs are thinned by electrolytic polishing. Electrolytic polishing technique is the window technique.<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">The sample is made the anode and a thin stainless sheet is made the cathode. The sample is immersed in the electrolyte, which is usually cooled by water or liquid nitrogen. Perchloric acid is usually used as the electrolyte. The sample edges are covered by lacquer to expose a &#8216;window&#8217;, hence the name. The experimental setup and the hole generation are shown in figure 8.<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-316\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167.png\" alt=\"\" width=\"347\" height=\"325\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167.png 347w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167-300x281.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167-65x61.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-167-225x211.png 225w\" sizes=\"auto, (max-width: 347px) 100vw, 347px\" \/><\/p>\n<p style=\"text-align: center\"><span style=\"text-align: initial;font-size: 1em\">Fig 8: Window polishing technique.<\/span><\/p>\n<div>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">When a current is applied the material is dissolved from the anode (sample) and deposits on the cathode. The rate of dissolution depends on the current and applied voltage. The I &#8211; V characteristics are shown in figure 9. Depending on the current and voltage, there are three regimes &#8211; etching, polishing, and pitting. The edges are coated so that material removal will start within the window. Once a hole is formed within the window the sample is pulled out. The region around the hole is usually electron transparent and can be mounted on a TEM grid.<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-317\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168.png\" alt=\"\" width=\"384\" height=\"233\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168.png 384w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168-300x182.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168-65x39.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168-225x137.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-168-350x212.png 350w\" sizes=\"auto, (max-width: 384px) 100vw, 384px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 9: I &#8211; V characteristics during polishing.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Ion milling technique:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">For non-conducting samples usually grinding and polishing steps are used in order to reduce sample thickness. Some an ultramicrotome is used in order to generate thin samples. These can be either electron transparent or can be used as the starting material for further thinning. The schematic of the technique is shown in Fig 10. For samples, where ultramicrotome cannot be used then a standard tripod polisher is used in order to thin the sample. This produces samples that are a few nm thick. The final polishing step is done by an ion beam miller.<\/p>\n<\/div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-318\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169.png\" alt=\"\" width=\"289\" height=\"461\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169.png 289w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169-188x300.png 188w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169-65x104.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-169-225x359.png 225w\" sizes=\"auto, (max-width: 289px) 100vw, 289px\" \/><\/p>\n<div>\n<p style=\"text-align: center\">Fig 10: Schematic of ultramicrotome technique.<\/p>\n<p>&nbsp;<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-319\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170.png\" alt=\"\" width=\"327\" height=\"419\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170.png 327w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170-234x300.png 234w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170-65x83.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-170-225x288.png 225w\" sizes=\"auto, (max-width: 327px) 100vw, 327px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">fig 11: The schematic of the ion beam miller and an actual instrument<\/p>\n<\/div>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">The sample is bombarded by high energy ions or neutral atoms. <\/span>Usually<span style=\"text-align: initial;font-size: 1em\"> Ar ions are used and they are formed by passing the Ar gas <\/span>though<span style=\"text-align: initial;font-size: 1em\"> a high voltage (4 &#8211; 6 keV ). The sample is held in <\/span>vacuum<span style=\"text-align: initial;font-size: 1em\"> and also usually cooled by liquid nitrogen. The ions are incident on the sample and sputter material away. To minimize ion penetration the beam is usually incident at a low angle (\u2248 20\u00b0) though if the angle is very small the sputter rate is small. <\/span>Ion<span style=\"text-align: initial;font-size: 1em\"> beam is highly controlled and a localized process but it is <\/span>time consuming<span style=\"text-align: initial;font-size: 1em\">. Sputter rates are usually a few A\u00b0 per second so that creating an electron transparent sample can take hours, especially if the initial thickness is high.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><strong style=\"text-align: initial;font-size: 1em\">Cross section sample preparation:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Slices from the sample are cut using a diamond slicer. These slices are placed between spacer layers and then glued on to a grid. The slices are glued in such a way that the interface is parallel to the slot in the grid. This sample is then thinned by standard tripod polishing until it is a few \u03bcm thick. The final sample is thinned using <\/span>a ion<span style=\"text-align: initial;font-size: 1em\"> beam miller to create an electron transparent sample.<\/span><\/p>\n<div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-320\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171.png\" alt=\"\" width=\"365\" height=\"266\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171.png 365w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171-300x219.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171-65x47.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171-225x164.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-171-350x255.png 350w\" sizes=\"auto, (max-width: 365px) 100vw, 365px\" \/><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: center\">Fig 12: Cross sectional sample preparation.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Replica technique:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: justify\">Replica technique is used for studying bulk specimens which cannot be destroyed to prepare electron specimens. It is also useful for studying surface topography features and precipitates though SEM techniques have gradually replaced replica sample preparation. A replica of the sample surface is prepared using a plastic mold. The mold is then removed from the surface and the surface of the specimen is replicated by the surface of the plastic. A thin film of carbon or metal like Cr, Pt is evaporated on the surface of the plastic. Sometimes the evaporation is done from an oblique angle, shadow evaporation, to enhance the contrast. The plastic is removed by dissolving in a suitable solvent and the film is then floated on to a grid for analysis.<\/p>\n<\/div>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-full wp-image-321\" src=\"http:\/\/msp08.epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172.png\" alt=\"\" width=\"533\" height=\"318\" srcset=\"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172.png 533w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172-300x179.png 300w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172-65x39.png 65w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172-225x134.png 225w, https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-content\/uploads\/sites\/109\/2018\/12\/Untitled-172-350x209.png 350w\" sizes=\"auto, (max-width: 533px) 100vw, 533px\" \/><\/p>\n<div>\n<p style=\"text-align: center\">Fig 13: Replica technique for sample preparation.<\/p>\n<\/div>\n<table>\n<tbody>\n<tr>\n<td><strong>you can view video on Transmission Electron Microscopy<\/strong><\/td>\n<td><a href=\"https:\/\/youtu.be\/PL3YdcGwOgE\" target=\"_blank\" rel=\"noopener\"><img loading=\"lazy\" decoding=\"async\" class=\"alignnone wp-image-120\" src=\"http:\/\/epgpbooks.inflibnet.ac.in\/wp-content\/uploads\/2018\/11\/download.png\" alt=\"\" width=\"36\" height=\"36\" \/><\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><strong style=\"text-align: initial;font-size: 1em\">References<\/strong><\/p>\n<ol>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">coen.boisestate.edu\/faculty-staff\/files\/2012\/01\/TEM.pdf<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Physical Principles of Electron Microscopy, Ray F. Egerton, Springer Verlag, 2007.<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Griffith G. (1993). Fine Structure Immunocytochemistry. New York, Berlin, Heidelberg. Springer Verlag. ISBN0-387-54805-X.<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron microscopy methods and <\/span>protocols \/ ed<span style=\"text-align: initial;font-size: 1em\">. by M.A. Nasser Hajibagheri. &#8211; Totowa, N.J. : Humana Press, cop. 1999. (Methods in molecular <\/span>biology ;<span style=\"text-align: initial;font-size: 1em\"> vol. 117)<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron <\/span>microscopy :<span style=\"text-align: initial;font-size: 1em\"> methods and protocols. &#8211; 2nd ed. \/ ed. by John Kuo &#8211; Totowa, N.J. : Humana Press, 2007. (Methods in molecular <\/span>biology ;<span style=\"text-align: initial;font-size: 1em\"> 369)<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Electron microscopy : principles and techniques for biologists \/ John J. Bozzola, Lonnie D.\u00a0<\/span><span style=\"text-align: initial;font-size: 1em\">Russell. &#8211; <\/span>Boston :<span style=\"text-align: initial;font-size: 1em\"> Jones and Bartlett, 1991. (The Jones and Bartlett series in biology)<\/span><\/li>\n<li style=\"text-align: justify\"><span style=\"text-align: initial;font-size: 1em\">Introduction to electron microscopy, Andres Kaech, April 2013.<\/span><\/li>\n<\/ol>\n<p><strong>\u00a0 \u00a0 Review your learning<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>1)\u00a0\u00a0\u00a0\u00a0\u00a0 Why are thin sections of specimens necessary in TEM?<\/p>\n<p>&nbsp;<\/p>\n<p>a)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons are negatively charged<\/p>\n<p>b)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have a wave nature<\/p>\n<p>c)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have no mass<\/p>\n<p>d)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons have a poor penetrating power.<\/p>\n<p>&nbsp;<\/p>\n<p>2)\u00a0\u00a0\u00a0\u00a0\u00a0 Why TEM images have much higher resolution than images from light microscope?<\/p>\n<p>&nbsp;<\/p>\n<p>a)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 TEM is much greater in size than light microscope<\/p>\n<p>b)\u00a0\u00a0\u00a0\u00a0\u00a0 Electrons travelling as waves have wavelength much shorter than visible light<\/p>\n<p>c)\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 TEM can achieve greater magnification<\/p>\n<p>d)\u00a0\u00a0\u00a0\u00a0\u00a0 The fluorescent screen of TEM can generate high resolution images<\/p>\n<p>&nbsp;<\/p>\n<p>3)\u00a0\u00a0\u00a0\u00a0\u00a0 Which of the following is the correct pathway of electrons in the TEM?<\/p>\n<p>&nbsp;<\/p>\n<p>a)\u00a0 \u00a0 \u00a0 Anode \u2192 electromagnetic lens system \u2192 sample \u2192 fluorescent screen<\/p>\n<p>b)\u00a0\u00a0\u00a0\u00a0\u00a0 Anode \u2192 electromagnetic lens system \u2192 sample \u2192 electromagnetic lens system \u2192 fluorescent screen<\/p>\n<p>c)\u00a0 \u00a0 \u00a0 Cathode \u2192 electromagnetic lens system \u2192 sample \u2192 electromagnetic lens system \u2192 fluorescent screen<\/p>\n<p>d)\u00a0\u00a0\u00a0\u00a0\u00a0 Cathode \u2192 electromagnetic lens system \u2192 sample \u2192 fluorescent screen<\/p>\n<p>&nbsp;<\/p>\n<p>4) What should be done right after the TEM column is shown to be evacuated?<\/p>\n<p>&nbsp;<\/p>\n<p>a) Insert the sample holder<\/p>\n<p>b) Further insert the sample holder<\/p>\n<p>c) Remove the dummy holder<\/p>\n<p>d) Shift the beam<\/p>\n<p>&nbsp;<\/p>\n<p>5) Before loading the sample, the following softwares have to be turned on, except\u2026<\/p>\n<p>&nbsp;<\/p>\n<p>a) Electron gun tilt\/shift<\/p>\n<p>b) TEM imaging and analysis<\/p>\n<p>c) Microscope user interface<\/p>\n<p>d) Digital Micrograph<\/p>\n<p>&nbsp;<\/p>\n<p><strong>True\/False:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>1) Ultra-violet light is applied to the sample to warm up the sample.<\/p>\n<p><span style=\"font-size: 1em\">2) The main purpose of cutting extremely thin slices of samples is for better observation of intracellular components instead of extracellular components.<\/span><\/p>\n<p><span style=\"font-size: 1em\">3) TEM cannot be used to examine <\/span>live<span style=\"font-size: 1em\"> specimen.<\/span><\/p>\n<p><span style=\"font-size: 1em\">4) We should turn off the light before <\/span>examination<span style=\"font-size: 1em\"> of <\/span>sample<span style=\"font-size: 1em\"> using the fluorescent screen.<\/span><\/p>\n<p><span style=\"font-size: 1em\">5) We need to do the alignment of the electron gun, beam and rotation center every time we use <\/span>the\u00a0<span style=\"font-size: 1em\">TEM<\/span><\/p>\n<p>&nbsp;<\/p>\n<p><strong>Fill in the Blanks:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>1)\u00a0\u00a0 The gun requires the vacuum of the order of\u00a0\u00a0 \u2026\u2026\u2026\u2026\u2026.<\/p>\n<p>2) The specimen requires the vacuum of the order of \u2026\u2026\u2026\u2026..<\/p>\n<p><span style=\"font-size: 1em\">3) Projection chamber and camera requires the vacuum of the order of \u2026\u2026<\/span><\/p>\n<p>&nbsp;<\/p>\n<p><strong>Long type questions:<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p>1) Explain the working principle of TEM<\/p>\n<p><span style=\"font-size: 1em\">2) Describe various types of pumps used to achieve vacuum?<\/span><\/p>\n","protected":false},"author":3,"menu_order":23,"template":"","meta":{"pb_show_title":"on","pb_short_title":"","pb_subtitle":"","pb_authors":["dr-s-s-islam"],"pb_section_license":""},"chapter-type":[],"contributor":[58],"license":[],"class_list":["post-303","chapter","type-chapter","status-publish","hentry","contributor-dr-s-s-islam"],"part":3,"_links":{"self":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapters\/303","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapters"}],"about":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/wp\/v2\/types\/chapter"}],"author":[{"embeddable":true,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/wp\/v2\/users\/3"}],"version-history":[{"count":5,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapters\/303\/revisions"}],"predecessor-version":[{"id":322,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapters\/303\/revisions\/322"}],"part":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/parts\/3"}],"metadata":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapters\/303\/metadata\/"}],"wp:attachment":[{"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/wp\/v2\/media?parent=303"}],"wp:term":[{"taxonomy":"chapter-type","embeddable":true,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/pressbooks\/v2\/chapter-type?post=303"},{"taxonomy":"contributor","embeddable":true,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/wp\/v2\/contributor?post=303"},{"taxonomy":"license","embeddable":true,"href":"https:\/\/ebooks.inflibnet.ac.in\/msp08\/wp-json\/wp\/v2\/license?post=303"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}