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Types of Electron Microscopes 1)Scanning Electron Microscope (SEM) The SEM is a microscope that uses electrons instead of light to form an image. The scanning electron microscope has many advantages over traditional microscopes. The SEM has a large depth of field, which allows more of a specimen to be in focus at one time. The SEM also has much higher resolution, so closely spaced specimens can be magnified at much higher levels. Because the SEM uses electromagnets rather than lenses, the researcher has much more control in the degree of magnification. All of these advantages, make the scanning electron microscope one of the most useful instruments in research today. In the SEM, we use much lower accelerating voltages to prevent beam penetration into the sample since what we require is generation of the secondary electrons from the true surface structure of a sample. Therefore, it is common to use low KV, in the range 1-5kV for biological samples, even though our SEMs are capable of up to 30 kV. How does a SEM work? A beam of electrons is produced at the top of the microscope by an electron gun. electron gun fitted with a tungsten filament cathode. Tungsten is normally used in electron guns because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission, and because of its low cost. The electron beam is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface. When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardropshaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 µm into the surface. The size of the interaction volume depends on the electron's landing energy, the atomic number of the specimen and the specimen's density. Once the beam hits the sample, electrons and X-rays are ejected from the sample. Detectors collect these X-rays, backscattered electrons, and secondary electrons and convert them into a signal that is sent to a screen similar to a television screen. This produces the final image. Scheme of Scanning electron microscopy Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) is a microscopy technique where by a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen. TEMs are capable of imaging at a significantly higher resolution than light microscopes. This enables the instrument's user to examine fine detail—even as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. TEMs find application in cancer research, virology, materials science as well as pollution, nanotechnology, and semiconductor research. At smaller magnifications TEM image contrast is due to absorption of electrons in the material, due to the thickness and composition of the material. At higher magnifications complex wave interactions modulate the intensity of the image, requiring expert analysis of observed images. Alternate modes of use allow for the TEM to observe modulations in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging. Source formation the TEM consists of an emission source, which may be a tungsten filament, or a lanthanum hexaboride (LaB6) source. or a small spikeshaped filament. LaB6 sources utilize small single crystals. By connecting this gun to a high voltage source (typically ~100–300 kV) the gun will, given sufficient current, begin to emit electrons either by field electron emission into the vacuum. Once extracted, the upper lenses of the TEM allow for the formation of the electron probe to the desired size and location for later interaction with the sample. the lens shape originating due to the distribution of magnetic flux. The lenses of a TEM allow for beam convergence, with the angle of convergence as a variable parameter, giving the TEM the ability to change magnification simply by modifying the amount of current that flows through the coil, quadrupole or hexapole lenses. The quadrupole lens is an arrangement of electromagnetic coils at the vertices of the square, enabling the generation of a lensing magnetic fields, the hexapole configuration simply enhances the lens symmetry by using six, rather than four coils. Typically a TEM consists of three stages of lensing. The stages are the condensor lenses, the objective lenses, and the projector lenses. The condensor lenses are responsible for primary beam formation, whilst the objective lenses focus the beam that comes through the sample itself. The projector lenses are used to expand the beam onto the phosphor screen or other imaging device, such as film. The magnification of the TEM is due to the ratio of the distances between the specimen and the objective lens' image plane . Display Imaging systems in a TEM consist of a phosphor screen, which may be made of fine (10–100 μm) particulate zinc sulphide, for direct observation by the operator. Optionally, an image recording system such as film based or doped YAG screen coupled . Typically these devices can be removed or inserted into the beam path by the operator as required. Components A TEM is composed of several components, which include a vacuum system in which the electrons travel, an electron emission source for generation of the electron stream, a series of electromagnetic lenses, as well as electrostatic plates. The latter two allow the operator to guide and manipulate the beam as required. Also required is a device to allow the insertion into, motion within, and removal of specimens from the beam path. Imaging devices are subsequently used to create an image from the electrons that exit the system. 1) Vacuum system To increase the mean free path of the electron gas interaction, a standard TEM is evacuated to low pressures, typically on the order of 10 −4 Pa. The need for this is twofold: first the allowance for the voltage difference between the cathode and the ground without generating an arc, and secondly to reduce the collision frequency of electrons with gas atoms to negligible levels—this effect is characterised by the mean free path. TEM components such as specimen holders and film cartridges must be routinely inserted or replaced requiring a system with the ability to reevacuate on a regular basis. As such, TEMs are equipped with multiple pumping systems and airlocks and are not permanently vacuum sealed. High-voltage TEMs require ultra-high vacuums on the range of 10−7 to 10−9 Pa to prevent generation of an electrical arc, particularly at the TEM cathode. Poor vacuum in a TEM can cause several problems, from deposition of gas inside the TEM onto the specimen as it is being viewed through a process known as electron beam induced deposition, or in more severe cases damage to the cathode from an electrical discharge. Vacuum problems due to specimen sublimation are limited by the use of a cold trap to adsorb sublimated gases in the vicinity of the specimen. 2) Specimen stage TEM specimen stage designs include airlocks to allow for insertion of the specimen holder into the vacuum with minimal increase in pressure in other areas of the microscope. The specimen holders are adapted to hold a standard size of grid upon which the sample is placed or a standard size of self-supporting specimen. Standard TEM grid sizes are a 3.05 mm diameter ring, with a thickness and mesh size ranging from a few to 100 μm. The sample is placed onto the inner meshed area having diameter of approximately 2.5 mm. Usual grid materials are copper, molybdenum, gold or platinum. This grid is placed into the sample holder, which is paired with the specimen stage. 3)Apertures فتحة، منفذ Apertures are annular metallic plates, through which electrons that are further than a fixed distance from the optic axis may be excluded. These consist of a small metallic disc that is sufficiently thick to prevent electrons from passing through the disc, whilst permitting axial electrons. This permission of central electrons in a TEM causes two effects simultaneously: firstly, apertures decrease the beam intensity as electrons are filtered from the beam, which may be desired in the case of beam sensitive samples. Secondly, this filtering removes electrons that are scattered to high angles, which may be due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample. Phase contrast Crystal structure can also be investigated by high-resolution transmission electron microscopy (HRTEM), also known as phase contrast. When utilizing a Field emission source and a specimen of uniform thickness, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. , the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex. However this effect can be used to an advantage, as it can be manipulated to provide more information about the sample, such as in complex phase retrieval techniques. What is the difference between SEM and TEM? In SEM (Scanning Electron Microscopy) you look at either backscattered or secondary electrones whereas in TEM (Transmission Electron Microscopy) you look how much of your electron beam makes it through the sample onto your phosphor screen or film camera. Usually SEM is used for surface analysis and TEM for analyzing sections. Both SEM (scanning electron microscope/microscopy) and TEM (transmission electron microscope/microscopy) refer both to the instrument and the method used in electron microscopy. There are a variety of similarities between the two. Both are types of electron microscopes and give the possibility of seeing, studying, and examining small, compositions of a sample. Both also use electrons (specifically, electron beams), the negative charge of an atom. Also, both samples in use are required to be “stained” or mixed with a particular element in order to produce images. Images produced from these instruments are highly magnified and have a high resolution. However, an SEM and TEM also share some differences. The method used in SEM is based on scattered electrons while TEM is based on transmitted electrons. The scattered electrons in SEM are classified as backscattered or secondary electrons. However, there is no other classification of electrons in TEM. The scattered electrons in SEM produced the image of the sample after the microscope collects and counts the scattered electrons. In TEM, electrons are directly pointed toward the sample. The electrons that pass through the sample are the parts that are illuminated in the image. The focus of analysis is also different. SEM focuses on the sample’s surface and its composition. On the other hand, TEM seeks to see what is inside or beyond the surface. SEM also shows the sample bit by bit while TEM shows the sample as a whole. SEM also provides a threedimensional image while TEM delivers a two-dimensional picture. In terms of magnification and resolution, TEM has an advantage compared to SEM. TEM has up to a 50 million magnification level while SEM only offers 2 million as a maximum level of magnification. The resolution of TEM is 0.5 angstroms while SEM has 0.4 nanometers. However, SEM images have a better depth of field compared to TEM produced images. Another point of difference is the sample thickness, “staining,” and preparations. The sample in TEM is cut thinner in contrast to a SEM sample. In addition, an SEM sample is “stained” by an element that captures the scattered electrons. On the other hand, TEM requires the sample to be prepared in a TEM grid and placed in the middle of the specialized chamber of the microscope. The image is produced by the microscope via fluorescent screens. Another feature of SEM is that the area where the sample is placed can be rotated in different angles. TEM was developed earlier than SEM. Disadvantages of Electron Microscopy Electron microscopes are very expensive to buy and maintain. They are dynamic rather than static in their operation: requiring extremely stable high voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high/ultra-high vacuum systems and a cooling water supply circulation through the lenses and pumps. As they are very sensitive to vibration and external magnetic fields, microscopes aimed at achieving high resolutions must be housed in buildings with special services. The samples have to be viewed in a vacuum, as the molecules that make up air would scatter the electrons. This means that the samples need to be specially prepared by sometimes lengthy and difficult techniques to withstand the environment inside an electron microscope. Recent advances have allowed some hydrated samples to be imaged using an environmental scanning electron microscope, but the applications for this type of imaging are still limited.