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The use of elevated gas pressures in the sample chamber of a scanning electron microscope (i.e., variable pressure SEM, or VPSEM) together with specialized electron detectors create imaging conditions that allow biological samples to be examined without any preparation. Specific operating conditions of elevated pressures combined with sample cooling (usually restricted to the environmental SEM range) can allow hydrated samples to be maintained in a pristine state for long periods of time. Dynamic processes also can be easily observed. A wider range of detector options and imaging parameters introduce greater complexity to the VPSEM operation than is present in routine SEM. The current instrumentation with field emission electron sources has nanometer-scale beam resolution (approx 1 nm) and low-voltage beam capability (0.1 kV). However, under the more extreme variable pressure conditions, useful biological sample information can be achieved by skilled operators at image resolutions to 2 to 4 nm and with primary electron beam voltages down to 1.0 kV. Imaging relating to electron charge behavior in some biological samples, generally referred to as charge contrast imaging, provides information unique to this VPSEM and environmental SEM that closely relates to luminescence imaged by confocal microscopy.
This chapter covers conventional methods for preparing biological specimens for examination in the scanning electron microscope (SEM). Techniques for handling cells grown in liquid culture, as well as on substrates such as culture dishes, slide culture chambers or agar, are discussed. These methods may be used to process most cultured organisms as well as whole botanical and zoological specimens.
1.Introduction and history of electron microscopes:-
o Electron microscopes are scientific instruments that use a beam of energetic electrons to examine objects on a very fine scale.
o Electron microscopes were developed due to the limitations of Light Microscopes which are limited by the physics of light.
o In the early 1930's this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria...etc.).
o This required 10,000x plus magnification which was not possible using current optical microscopes.
The rate of a reaction involving heterogeneous
2.What are Electron Microscopes?
Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the following information:
The surface features of an object or "how it looks", its texture; direct relation between these features and materials properties (hardness, reflectivity...etc.)
The shape and size of the particles making up the object; direct relation between these structures and materials properties (ductility, strength, reactivity...etc.)
The elements and compounds that the object is composed of and the relative amounts of them; direct relationship between composition and materials properties (melting point, reactivity, hardness...etc.)
How the atoms are arranged in the object; direct relation between these arrangements and materials properties (conductivity, electrical properties, strength...etc.)
3.How do Electron Microscopes Work?
Electron Microscopes(EMs) function exactly as their optical counterparts except that they use a focused beam of electrons instead of light to "image" the specimen and gain information as to its structure and composition. The basic steps involved in all EMs:
A stream of electrons is formed (by the Electron Source) and accelerated toward the specimen using a positive electrical potential This stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam. This beam is focused onto the sample using a magnetic lens Interactions occur inside the irradiated sample, affecting the electron beam These interactions and effects are detected and transformed into an image The above steps are carried out in all EMs regardless of type. A more specific treatment of the workings of two different types of EMs are described in more detail:
4. Introduction of Scanning Electron Microscopy(SEM):-
The rate of a reaction involving heterogeneous catalysis is a measure of the activity of the catalyst . While chemisorption phenomena,surface area, pore volume, pore size distribution and surface topography affect activity, Taylor has proposed that certain "active sites", representing a small fraction of the total surface area, are responsible for catalytic activity. Numerous studies carried out to identify these active sites have been concerned with crystal structure and with morphological features such as lattice defects, dislocations, and surface steps. Employing transmission electron micro- scopy, electron and X-ray diffraction, these studies, carried out on highly pure materials, preferably well-defined single crystals, have shown that catalytic activity can be correlated with surface morphology. Although parallel studies on complex systems such as polycrystal- line, multicomponent catalysts of the type frequently employed in technical processes have not been carried out, we may assume that here too there is a direct relationship between surface morphology and catalytic activity. Studies employing indirect methods, mostly those based on adsorption phenomena have been carried out on these complex systems and show in many cases a direct relationship between surface area, pore size, and pore volume on the one hand, and catalytic activity on the other.
The scanning electron microscope (SEM), which has recently become available, permits a direct examination of the surfaces of complex catalyst systems. Using scanning electron micro- scopy, the effects of processing conditions on surface morphology can conveniently be studied and predictions on the performance of a catalyst made, if one accepts the premise that there exists a direct relationship between changes in the microstructure of catalyst surfaces and changes in the morphology resolvable with the SEM.
Scanning Electron Microscope (SEM):-
SEMs are patterned after Reflecting Light Microscopes and yield similar information:
The surface features of an object or "how it looks", its texture; detectable features limited to a few manometers
The shape, size and arrangement of the particles making up the object that are lying on the surface of the sample or have been exposed by grinding or chemical etching; detectable features limited to a few manometers
The elements and compounds the sample is composed of and their relative ratios, in areas ~ 1 micrometer in diameter
The arrangement of atoms in the specimen and their degree of order; only useful on single-crystal particles >20 micrometers
A detailed explanation of how a typical SEM functions follows (refer to the diagram below): -
The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons.
The stream is condensed by the first condenser lens (usually controlled by the "coarse probe current knob"). This lens is used to both form the beam and limit the amount of current in the beam. It works in conjunction with the condenser aperture to eliminate the high-angle electrons from the beam
The beam is then constricted by the condenser aperture (usually not user selectable), eliminating some high-angle electrons
The second condenser lens forms the electrons into a thin, tight, coherent beam and is usually controlled by the "fine probe current knob"
A user selectable objective aperture further eliminates high-angle electrons from the beam
A set of coils then "scan" or "sweep" the beam in a grid fashion (like a television), dwelling on points for a period of time determined by the scan speed (usually in the microsecond range)
The final lens, the Objective, focuses the scanning beam onto the part of the specimen desired.
When the beam strikes the sample (and dwells for a few microseconds) interactions occur inside the sample and are detected with various instruments
Before the beam moves to its next dwell point these instruments count the number of interactions and display a pixel on a CRT whose intensity is determined by this number (the more reactions the brighter the pixel).
This process is repeated until the grid scan is finished and then repeated, the entire pattern can be scanned 30 times per second.
elow are shown various examples of the type of results obtainable using the Facility's SEM. Click on the image you want to download to receive a larger version (in JPEG format with the size in brackets). If you wish to save a copy of the image be sure to change your browser's mode to "Load to Disk" so you can save the image as a file.
Mechanically Alloyed Cu-Nb-Fe Organically Deposited Iron Oxide
Fracture Surface of Paper Clip Brittle Fracture of Steel
The scanning electron microscope (SEM) is a type of electron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity.
The types of signals produced by an SEM include secondary electrons, back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen current and transmitted electrons. Secondary electron detectors are common in all SEMs, but it is rare that a single machine would have detectors for all possible signals. The signals result from interactions of the electron beam with atoms at or near the surface of the sample. In the most common or standard detection mode, secondary electron imaging or SEI, the SEM can produce very high-resolution images of a sample surface, revealing details about less than 1 to 5 nm in size. Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. This is exemplified by the micrograph of pollen shown to the right. A wide range of magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the magnification limit of the best light microscopes. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. BSE are often used in analytical SEM along with the spectra made from the characteristic X-rays. Because the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen, BSE images can provide information about the distribution of different elements in the sample. For the same reason, BSE imaging can image colloidal gold immuno-labels of 5 or 10 nm diameter which would otherwise be difficult or impossible to detect in secondary electron images in biological specimens. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher energy electron to fill the shell and release energy. These characteristic X-rays are used to identify the composition and measure the abundance of elements in the sample.
5.Scanning process and image formation:-
Schematic diagram of an SEM.
In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic 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. Other types of electron emitters include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and field emission guns (FEG), which may be of the cold-cathode type using tungsten single crystal emitters or the thermally-assisted Schottky type, using emitters of zirconium oxide.
The electron beam, which typically has an energy ranging from 0.5 keV to 40 keV, 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 teardrop-shaped 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. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types are used to amplify the signals which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image may be captured by photography from a high resolution cathode ray tube, but in modern machines is digitally captured and displayed on a computer monitor and saved to a computer's hard disk.
6.Advantages of Using SEM over OM :-
Mag Depth of Field Resolution
OM: 4x - 1400x 0.5mm ~ 0.2mm
SEM: 10x - 500Kx 30mm 1.5nm
The SEM has a large depth of field, which allows a large amount of the sample to be in focus at one time and produces an image that is a good representation of the three-dimensional sample.
The combination of higher magnification, larger depth of field, greater resolution, compositional and crystallographic information makes the SEM one of the most heavily used instruments in academic/national lab research areas and industry.
Conventional SEM requires samples to be imaged under vacuum, because a gas atmosphere rapidly spreads and attenuates electron beams. Consequently, samples that produce a significant amount of vapour, e.g. wet biological samples or oil-bearing rock need to be either dried or cryogenically frozen. Processes involving phase transitions, such as the drying of adhesives or melting of alloys, liquid transport, chemical reactions, solid-air-gas systems in general cannot be observed. Some observation of live samples has been possible 
The first commercial development of the Environmental SEM (ESEM) in the late 1980s HYPERLINK "http://en.wikipedia.org/wiki/Scanning_electron_microscope#cite_note-20" allowed samples to be observed in low-pressure gaseous environments (e.g. 1-50 Torr) and high relative humidity (up to 100%). This was made possible by the development of a secondary-electron detector capable of operating in the presence of water vapour and by the use of pressure-limiting apertures with differential pumping in the path of the electron beam to separate the vacuum region (around the gun and lenses) from the sample chamber.
The first commercial ESEMs were produced by the ElectroScan Corporation in USA in 1988. ElectroScan were later taken over by Philips (who later sold their electron-optics division to FEI Company) in 1996 .
ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Uncoated Plastics and Elastomers can be routinely examined, as can uncoated biological samples. Coating can be difficult to reverse, may conceal small features on the surface of the sample and may reduce the value of the results obtained. X-ray analysis is difficult with a coating of a heavy metal, so carbon coatings are routinely used in conventional SEMs, but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens. ESEM may be the preferred for electron microscopy of unique samples from criminal or civil actions, where forensic analysis may need to be repeated by several different experts.