An Electronically Magnified Image Of A Specimen Biology Essay


An electron microscope is a type of microscope that produces an electronically-magnified image of a specimen for detailed observation. The electron microscope (EM) uses a particle beam of electrons to illuminate the specimen and create a magnified image of it. The microscope has a greater resolving power than a light-powered optical microscope, because it uses electrons that have wavelengths about 100,000 times shorter than visible light (photons), and can achieve magnifications of up to 2,000,000x, whereas light microscopes are limited to 2000x magnification.

The electron microscope uses electrostatic and electromagnetic "lenses" to control the electron beam and focus it to form an image. These lenses are analogous to, but different from the glass lenses of an optical microscope that form a magnified image by focusing light on or through the specimen.

Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is primarily used for quality control and failure analysis in semiconductor device fabrication.

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The transmission electron microscope (TEM) is a scientific instrument that uses electrons instead of light to scrutinize objects at very fine resolutions. They were developed in the 1930s when scientists realized that electrons can be used instead of light to "magnify" objects or specimens under study

Transmission electron microscopy (TEM)

is a microscopy technique whereby 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; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.

TEMs are capable of imaging at a significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons. 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 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


The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first commercial TEM in 1939

In 1931, the German physicist Ernst Ruska and German electrical engineer Max Knoll constructed the prototype electron microscope, capable of four-hundred-power magnification; the apparatus was a practical application of the principles of electron microscopy. Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (lens) microscope.]Moreover, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained the patent for the electron microscope in May 1931. Family illness compelled the electrical engineer to devise an electrostatic microscope, because he wanted to make visible the poliomyelitis virus.

In 1937, the Siemens company financed the development work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska (Ernst's brother) to develop applications for the microscope, especially with biologic specimens. Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope. The first practical electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus; and Siemens produced the first commercial Transmission Electron Microscope (TEM) in 1939. Although contemporary electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska's prototype.


Transmission electron microscope (TEM):-

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The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image. The electrons are emitted by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic andelectromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") is viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide. The image can be photographically recorded by exposing a photographic film or platedirectly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed on a monitor or computer.

Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the High Resolution TEM (HRTEM) has allowed the production of images with resolution below 0.5 Ångström (50 picometres) at magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.

Scanning electron microscope (SEM):-

Unlike the TEM, where electrons of the high voltage beam carry the image of the specimen, the electron beam of the Scanning Electron Microscope (SEM) does not at any time carry a complete image of the specimen. The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). At each point on the specimen the incident electron beam loses some energy, and that lost energy is converted into other forms, such as heat, emission of low-energy secondary electrons, light emission (cathodoluminescence) or x-ray emission. The display of the SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown at right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs.

Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three-dimensional shape of the sample.

Reflection electron microscope (REM):-

In the Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam ofelastically scattered electrons is detected. This technique is typically coupled with Reflection High Energy Electron Diffraction (RHEED) and Reflection high-energy loss spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains.

Scanning transmission electron microscope (STEM):-

The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifiesannular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion.

Low voltage electron microscope (LVEM):-

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The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which operates at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which is especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the need to stain. Sectioned samples generally need to be thinner than they would be for conventional TEM (20-65 nm). Resolutions of a few nm are possible in TEM, SEM and STEM modes.

Initial development:-

Ernst Abbe originally proposed that the ability to resolve detail in an object was limited by thewavelength of the light used in imaging, thus limiting the useful obtainable magnification from an optical microscope to a few micrometers. Developments into ultraviolet (UV) microscopes, led byKoehler, allowed for an increase in resolving power of about a factor of two. However this required more expensive quartz optical components, due to the absorption of UV by glass. At this point it was believed that obtaining an image with sub-micrometer information was simply impossible due to this wavelength constraint.

It had earlier been recognized by Plücker in 1858 that the deflection of "cathode rays" (electrons) was possible by the use of magnetic fields. This effect had been utilised to build primitive cathode ray oscilloscopes (CROs) as early as 1897 by Ferdinand Braun, intended as a measurement device. Indeed in 1891 it was recognized by Riecke that the cathode rays could be focused by these magnetic fields, allowing for simple lens designs. Later this theory was extended by Hans Busch in his work published in 1926, who showed that the lens maker's equation, could under appropriate assumptions, be applicable to electrons.

In 1928, at the Technological University of Berlin Adolf Matthias, Professor of High voltage Technology and Electrical Installations, appointed Max Knoll to lead a team of researchers to advance the CRO design.


Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope systems now overlaps with those of basic electron microscopes. 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- or 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 designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems. Some desktop low voltage electron microscopes have TEM capabilities at very low voltages (around 5 kV) without stringent voltage supply, lens coil current, cooling water or vibration isolation requirements and as such are much less expensive to buy and far easier to install and maintain, but do not have the same ultra-high (atomic scale) resolution capabilities as the larger instruments.

The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr/2.7 kPa), wet environment.

Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; however, this process has the potential to disturb delicate samples.

entists, further confirming the validity of this technique.


The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons.

This stream is focused to a small, thin, coherent beam by the use of condenser lenses 1 and 2. The first lens(usually controlled by the "spot size knob") largely determines the "spot size"; the general size range of the final spot that strikes the sample. The second lens(usually controlled by the "intensity or brightness knob" actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.

The beam is restricted by the condenser aperture (usually user selectable), knocking out high angle electrons (those far from the optic axis, the dotted line down the center)

The beam strikes the specimen and parts of it are transmitted

This transmitted portion is focused by the objective lens into an image

Optional Objective and Selected Area metal apertures can restrict the beam; the Objective aperture enhancing contrast by blocking out high-angle diffracted electrons, the Selected Area aperture enabling the user to examine the periodic diffraction of electrons by ordered arrangements of atoms in the sample

The image is passed down the column through the intermediate and projector lenses, being enlarged all the way

The image strikes the phosphor image screen and light is generated, allowing the user to see the image. The darker areas of the image represent those areas of the sample that fewer electrons were transmitted through (they are thicker or denser). The lighter areas of the image represent those areas of the sample that more electrons were transmitted through (they are thinner or less dense)

Applications of the Transmission ElectronMicroscope:-

The TEM has its primary uses in metallurgy (or the study of metals and minerals) and the biological sciences, especially in the study of cells at the molecular level. TEMs have been particularly useful in metallurgy, especially in terms of developing images of crystals and metals at the molecular level - allowing scientist scientists to study their structure, interactions and identify flaws.

The downside to the TEM lies in the specimens that can be studied - these have to be 'sliced' very, very thinly to ensure that they are 'electron transparent'; they must also be placed in a vacuum. As such, preparation of specimens is often very time consuming and requires expert handling. This leads to concerns that specimens prepared for TEM study will be inadvertently damaged in the process. This raises the question of whether the specimen is as pure as can be expected. Finally, there are also concerns that the bombardment of electrons may damage the specimen under scrutiny - especially if these are biological samples.


An old-fashioned slide projector works by projecting light through a film layer. As the light passes through the film, it interacts with the film and specific areas of the film lets light pass through unobstructed, other areas absorb light and doesn't let it pass through, and still some absorb part of the light and lets only a fraction get through. The light that does go through is hits the lenses found on the other side and the resulting image is projected onto a screen.

The TEM works similarly. In the case of the TEM, though, a beam of electrons are focused on a single, pinpoint spot or element on the sample being studied. The electrons interact with the sample and only those that go past unobstructed hit the phosphor screen on the other side. At this point, the electrons are converted to light and an image is formed.

The dark areas of the image correspond to areas on the specimen where fewer electrons were able to pass through (either absorbed or scattered upon impact); the lighter areas are where more electrons did pass through, although the varying amounts of electrons in these areas enable the user to see structures and gradients.

The 'lenses' in a TEM are not the same as lenses in a conventional microscope; these are actually EM devices that can 'focus' the electron beam to the desired wavelength or size. In much the same way as a light microscope, however, the amount of power used to generate electrons allows for higher magnification or better resolutions.

Principles of operation:-

The transmission electron microscope uses a high energy electron beam transmitted through a very thin sample to image and analyze the microstructure of materials with atomic scale resolution. The electrons are focused with electromagnetic lenses and the image is observed on a fluorescent screen, or recorded on film or digital camera. The electrons are accelerated at several hundred kV, giving wavelengths much smaller than that of light: 200kV electrons have a wavelength of 0.025Å. However, whereas the resolution of the optical microscope is limited by the wavelength of light, that of the electron microscope is limited by aberrations inherent in electromagnetic lenses, to about 1-2 Å.

Because even for very thin samples one is looking through many atoms, one does not usually see individual atoms. Rather the high resolution imaging mode of the microscope images the crystal lattice of a material as an interference pattern between the transmitted and diffracted beams. This allows one to observe planar and line defects, grain boundaries, interfaces, etc. with atomic scale resolution. The brightfield/darkfield imaging modes of the microscope, which operate at intermediate magnification, combined with electron diffraction, are also invaluable for giving information about the morphology, crystal phases, and defects in a material. Finally the microscope is equipped with a special imaging lens allowing for the observation of micromagnetic domain structures in a field-free environment.

The TEM is also capable of forming a focused electron probe, as small as 20 Å, which can be positioned on very fine features in the sample for microdiffraction information or analysis of x-rays for compositional information. The latter is the same signal as that used for EMPA and SEM composition analysis , where the resolution is on the order of one micron due to beam spreading in the bulk sample. The spatial resolution for this compositional analysis in TEM is much higher, on the order of the probe size, because the sample is so thin. Conversely the signal is much smaller and therefore less quantitative. The high brightness field-emission gun improves the sensitivity and resolution of x-ray compositional analysis over that available with more traditional thermionic sources.


The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. An electron cryomicroscope (CryoTEM) is a TEM with a specimen holder capable of maintaining the specimen at liquid nitrogen or liquid helium temperatures. This allows imaging specimens prepared in vitreous ice, the preferred preparation technique for imaging individual molecules or macromolecular assemblies.

A TEM can be modified into a scanning transmission electron microscope (STEM) by the addition of a system that rasters the beam across the sample to form the image, combined with suitable detectors. Scanning coils are used to deflect the beam, such as by an electrostatic shift of the beam, where the beam is then collected using a current detector such as a faraday cup, which acts as a direct electron counter. By correlating the electron count to the position of the scanning beam (known as the "probe"), the transmitted component of the beam may be measured. The non-transmitted components may be obtained either by beam tilting or by the use of annular dark field detectors.


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.

 Vacumm 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 re-evacuate on a regular basis. As such, TEMs are equipped with multiple pumping systems and airlocks and are not permanently vacuum sealed.

The vacuum system for evacuating a TEM to an operating pressure level consists of several stages. Initially a low or roughing vacuum is achieved with either a rotary vane pump or diaphragm pumps bringing the TEM to a sufficiently low pressure to allow the operation of a turbomolecular or diffusion pump which brings the TEM to its high vacuum level necessary for operations. To allow for the low vacuum pump to not require continuous operation, while continually operating the turbomolecular pumps, the vacuum side of a low-pressure pump may be connected to chambers which accommodate the exhaust gases from the turbomolecular pump. Sections of the TEM may be isolated by the use of gate valves, to allow for different vacuum levels in specific areas, such as a higher vacuum of 10−4 to 10−7 Pa or higher in the electron gun in high resolution or field emission TEMs.

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. As such for higher voltage TEMs a third vacuum system may operate, with the gun isolated from the main chamber either by use of gate valves or by the use of a differential pumping aperture. The differential pumping aperture is a small hole that prevents diffusion of gas molecules into the higher vacuum gun area faster than they can be pumped out. For these very low pressures either an ion pump or a getter material is used.

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.


With the development of TEM, the associated technique of scanning transmission electron microscopy (STEM) was re-investigated and did not become developed until the 1970s, withAlbert Crewe at the University of Chicago developing the field emission gun] and adding a high quality objective lens to create the modern STEM. Using this design, Crewe demonstrated the ability to image atoms using annular dark-field imaging. Crewe and coworkers at the University of Chicago developed the cold field electron emission source and built a STEM able to visualize single heavy atoms on thin carbon substrates.