The Transmission Electron Microscopy Biology Essay
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Published: Mon, 5 Dec 2016
The transmission electron microscope operates on the same basic principles as the light microscope but uses electrons instead of light. What you can see with a light microscope is limited by the wavelength of light. TEMs use electrons as “light source” and their much lower wavelength make it possible to get a resolution a thousand times better than with a light microscope. TEM uses a 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, nanotechnology, and semiconductor research.
History of TEMs
The first operational electron microscope was presented by Ernst Ruska and Max Knoll in 1932, and 6 years later Ruska had a first version on the market. In 1986 Ruska received a Nobel Prize in physics for his “fundamental work in electron optics and for the design of the first electron microscope”. The following table gives a basic outline of the history of the electron microscope by decades.
-50kV, single condenser
-little or no theory; a first basic theory of electron microscopy was published in 1949 by Heidenreich.
-contrast theory developed.
Dynamic in-situ studies
substructure of solids
-high voltage electron microscopes (Toulouse: 1.2 and 3MeV)
-scanning electron microscopes
-accessories for in-situ studies
High resolution imaging
-Analytical transmission electron microscopy
-scanning transmission electron microscopy
-energy dispersive x-ray spectra
-electron energy loss spectroscopy
-commercial high voltage electron microscopy (0.4-1.5MeV)
-high resolution imaging theory
7nm (standard scanning)
virtually all materials
atomic resolution in close-packed solids
-commercial medium-voltage high-resolution/analytical electron microscopy (300-400kV)
-improved analytical capabilities
-energy filtering imaging
-ultra-high vacuum microscopes
5nm (scanning at 1kV)
fast computation for image simulation
integrated digital scanning and image processing
-surface atomic microscopy
-orientation imaging microscopy
3nm (scanning at 1kV)
Electron microscopy in the 1960s
In 1969 RCA dropped out of the electron microscope business, having decided that they could make more money selling record albums and consumer electronic devices. General Electric had never become a major power in the electron microscope business. This left the field wide open for companies such as JEOL, Hitachi, and Akashi in Japan, and Philips, Siemens, and Zeiss in Europe.
The resolution of the best TEMs was now approximately 0.3 nm (3 Å); JEOL claimed a resolution of 0.2 nm (2 Å) for its 1968 model JEM-100B. Accelerating voltages were still typically in the 100 kV range, although JEOL marketed a 200 kV instrument in 1967 called the JEM-200. Philips marketed a very popular 100 kV microscope called the EM 300 in 1966. They claimed that this was the ‘first fully-transistorized electron microscope,’ and that it could attain a point resolution of 0.5 nm (5 Å). More than 1,850 units of the EM 300 were sold.
Another approach to the study of materials that emerged in the 1960s involved increasing the accelerating voltage of the electron gun to extreme levels up to 3 MeV in an effort to penetrate more deeply into thicker samples. CEMES-LOE/CNRS at Toulouse, France, developed a 3MeV instrument around 1965, followed closely by JEOL, which released a 1 MeV microscope, the JEM-1000, in 1966. (One MeV represents a million electron volts, while one kV is a thousand electron volts. So 1,000 kV= 1 MeV.)
These ultrahigh voltage EMs were so large that they typically occupied their own two-story building. The electron gun and its associated high voltage electronics were located near the ceiling of the second story, while the operator sat at the bottom of the microscope column looking at the fluorescent screen. Hitachi’s 1964 model HU-500 stood 4 meters tall; later, higher MeV versions eventually made this look small. On the left is a photograph of the 1 MeV Atomic Resolution Microscope (ARM) at the Lawrence Berkeley Laboratory.
Electron microscopy in the 1970s
The 1970s were a time of rapid development on all fronts in the electron microscope industry. Further improvements in TEM came from brighter electron sources (lanthanum hexaboride and field emission guns).
The resolution of the TEM was pushed to 0.2 nm (2 Å) in the 1970s, with better results reported in some cases for lattice imaging resolutions; Hitachi claimed a 1.4 Å lattice resolution for its 1975 model H-500 TEM, and JEOL claimed the same resolution for its 1973 model JEM-100C. Accelerating voltages of 100 kV maximum had become the norm.
In contrast to the low cost instruments, Philips 1972 model EM 301 TEM was designed for high performance and versatility for the skilled operator who had the time to coax the best results from his instrument. The EM 400 introduced in 1975 used a LAB6 electron gun, which was ten times as bright as the standard tungsten filament at the time. On the down side, the reactivity of lanthanum hexaboride required an ultra-clean vacuum system of 10-6 Torr. In 1977 Philips introduced accessories for the EM 400, including a secondary electron detector for topographical studies and a field emission gun (FEG) a single crystal tungsten tipped filament that emits electrons from a very localized region of the tip to produce narrow, bright electron beams. FEGs can have100 to 1,000 times the brightness of a LAB6 filament, with electron beam diameters as small as 1 nm. Vacuum requirements for these FEGs are 10-10 Torr.
JEOL started with the JEM-100B Analytical model in 1970, which added scanning ability and an EDX x-ray spectrometer to the TEM. This was improved upon by the JEM-100C in 1973, with its 1.4 Å resolution, and further upgraded by the JEM-100CX Analytical model in 1976, which added an ultraclean vacuum system and a LAB6 electron gun.
In the ultrahigh voltage EM market, The Hitachi 3MeV HU-3000 was installed at Osaka University in 1970. This accelerating voltage was the highest ever for an electron microscope. A resolution of 4.6 Å was reported for this instrument. The 1976 model H-1250 had a maximum voltage of 1250 kV, but a superior resolution of 2.04 Å.
Electron microscopy in the 1980s
During the 1980s TEM resolutions were further reduced to 1.0 to 1.5Å, making imaging of atoms in lattice planes possible. Microprocessor control of microscopes and computerized analysis of data became common due to the emergence of the personal computer in the early 80s. This microprocessor control brought about such features as an auto-stigmator and auto-focus, freeing the microscope operator from the mundane tasks that had always been involved in using the instrument. Electron energy loss spectroscopy (EELS) detectors were incorporated in STEMs and AEMs, allowing detection of low atomic number elements that could not be seen using x-ray techniques. The demands of the fast-growing integrated circuits industry produced electron microscopes designed for non-destructive testing of semiconductor wafers and for functional testing of ICs. Smaller electron beam sizes made it possible to switch from microprobe to nanoprobe technology. Elemental mapping of a sample’s surface could now be done on a nanometer level.
Development of low cost instruments was not a priority in the 1980s. Some that were developed in the 1970s continued to be sold, but development was focused on high-performance, high-resolution, microprocessor-controlled instruments.
JEOL produced 7 new TEM units between 1980 and 1986. These included the JEM-1200 EX (1981), which added microprocessor control to the JEM-100 CX (1976). The same model equipped with an EDS x-ray spectrometer was called the JEM-1200 EX/Analytical microscope. The 1984 model JEM-2000 FX/Analytical had a maximum voltage of 200 kV and a resolution of 2.8 Å; this instrument marked the switch from a microprobe beam to a nanoprobe. The JEM-4000 FX/Analytical microscope introduced in 1986 raised the acceleration voltage to 400 kV, which produced a beam probe size only 2 nm in diameter. After years of a standard 100 kV accelerating voltage with a few ultrahigh voltage units thrown in, these medium-voltage microscopes finally became popular.
Electron microscopy in the 1990s
The 1990s produced several corporate mergers in the electron microscope industry. Carl Zeiss and Leica joined to form LEO Electron Microscopy, Inc. In 1996 Philips bought Electroscan, the developer of the environmental SEM in the 1980s, to form Philips Electroscan. The following year Philips Electron Optics and a company called FEI merged under the name FEI to continue manufacturing electron microscopes. Hitachi and JEOL remained independent entities.
The resolution of TEMs had already reached its theoretical limit (the best possible resolution predicted by calculations), so the 1Å resolution obtained using field emission gun (FEG) electron sources remained the standard. Medium voltage range instruments up to 300 kV were common, although 100 kV instruments still kept their long lasting popularity.
Computers were now a vital part of every electron microscope, with graphical user interfaces (GUIs) being the norm. They were involved in both the control of the instrument and the processing of data, including post-analysis enhancement of micrographs using contrast-enhancing software.
JEOL offered TEMs with maximum accelerating voltages of 120, 200, and 300 kV. The 120 kV model JEM1230 had a resolution of 0.2 nm (2Å). The JEM-2010 F FasTEM (200 kV) and the JEM-3000 F FasTEM (300 kV) both used FEG sources and achieved resolutions of 0.1 nm (1.0 Å).
Three meetings of the Electron Microscopy Society of America (1968, 1975, and 1980)
The Electron Microscopy Society of America (now known as the Microscopy Society of America) was founded in 1942, when it began holding annual meetings for instrument makers and users to gather and discuss the technology and its applications. The topics of papers given at these meetings present a snapshot of the state of electron microscopy at the time. A brief look at three of these meetings shows the evolution of the technology and its applications over a 12-year period.
In the brief twelve-year span of 1968 to 1980, the physical sciences overtook the biological sciences at EMSA meetings, judging solely on number of papers presented. A large part of this development is probably due to the emergence of the scanning electron microscope in 1965, which made examination of the surface of bulk specimens possible for the first time. Since physical scientists could now look at “real” samples instead of replicas or thin films, activity in microscopy of materials increased dramatically. With no similar dramatic development in biological microscopy, the balance shifted.
The Science of TEMs
Comparison of Light (LM) and Electron Microscopes.
1) Illumination system: produces required radiation and directs it onto the specimen. Consists of a source, which emits the radiation, and a condenser lens, which focuses the illuminating beam (allowing variations of intensity to be made) on the specimen.
2) Specimen stage: situated between the illumination and imaging systems.
3) Imaging system: Lenses which together produce the final magnified image of the specimen. Consists of i) an objective lens which focuses the beam after it passes through the specimen and forms an intermediate image of the specimen and ii) the projector lens(es) which magnifies a portion of the intermediate image to form the final image.
4) Image recording system: Converts the radiation into a permanent image (typically on a photographic emulsion) that can be viewed.
1) Optical lenses are generally made of glass with fixed focal lengths whereas magnetic lenses are constructed with ferromagnetic materials and windings of copper wire producing a focal length which can be changed by varying the current through the coil.
2) Magnification in the LM is generally changed by switching between different power objective lenses mounted on a rotating turret above the specimen. It can also be changed if oculars (eyepieces) of different power are used. In the TEM the magnification (focal length) of the objective remains fixed while the focal length of the projector lens is changed to vary magnification.
3) The LM has a small depth of field, thus different focal levels can be seen in the specimen. The large (relative) depth of field in the TEM means that the entire (thin) specimen is in focus simultaneously.
4) Mechanisms of image formation vary (phase and amplitude contrast).
5) TEMs are generally constructed with the radiation source at the top of the instrument: the source is generally situated at the bottom of LMs.
6) TEM is operated at high vacuum (since the mean free path of electrons in air is very small) so most specimens (biological) must be dehydrated.
7) TEM specimens (biological) are rapidly damaged by the electron beam.
8) TEMs can achieve higher magnification and better resolution than LMs.
9) Price tag!!! (100x more than LM)
Figure below shows the cross-sectional view of a standard TEM.
Figure shows the transmission electron microscope at The Chinese University of Hong Kong.
Figure shows a schematic outline of a TEM. A TEM contains four parts: electron source, electromagnetic lens system, sample holder, and imaging system.
A. Electron Source
The electron gun produces a beam of electrons whose kinetic energy is high enough to enable them to pass through thin areas of the TEM specimen. The gun consists of an electron source, also known as the cathode because it is at a high negative potential, and an electron-accelerating chamber. There are several types of electron source, operating on different physical principles, which we now discuss.
i. Thermionic Emission
Figure 3-1 shows a common form of electron gun. The electron source is a V-shaped (“hairpin”) filament made of tungsten (W) wire, spot-welded to straight-wire leads that are mounted in a ceramic or glass socket, allowing the filament assembly to be exchanged easily when the filament eventually “burns out.” A direct (dc) current heats the filament to about 2700 K, at which temperature tungsten emits electrons into the surrounding vacuum by the process known as thermionic emission.
Thermionic electron gun containing a tungsten filament F, Wehnelt electrode W, ceramic high-voltage insulator C, and o-ring seal O to the lower part of the TEM column. An autobias resistor, RB (actually located inside the high-voltage generator, as in Fig. 3-6) is used to generate a potential difference between W and F; thereby controlling the electron-emission current, Ie. Arrows denote the direction of electron flow that gives rise to the emission current.
Raising the temperature of the cathode causes the nuclei of its atoms to vibrate with increased amplitude. Because the conduction electrons are in thermodynamic equilibrium with the atoms, they share this thermal energy, and a small proportion of them achieve energies above the vacuum level, enabling them to escape across the metal/vacuum interface.
The rate of electron emission can be represented as a current density Je(in A/m2) at the cathode surface, which is given by the Richardson law:
Where T is the absolute temperature (in K) of the cathode and A is the Richardson constant (~106Am-2K-2), which depends to some degree on the cathode material but not on its temperature; k is the Boltzmann constant (1.38 x 10-23J/K), and kT is approximately the mean thermal energy of an atom.
ii. Schottky emission
The thermionic emission of electrons can be increased by applying an electrostatic field to the cathode surface. This field lowers the height of the potential barrier (which keeps electrons inside the cathode) by an amount, the so-called Schottky effect.
A Schottky source consists of a pointed crystal of tungsten welded to the end of V-shaped tungsten filament. The tip is coated with zirconium oxide (ZrO) to provide a low work function (~2.8 eV) and needs to be heated to only about 1800 K to provide adequate electron emission. Because the tip is very sharp, electrons are emitted from a very small area, resulting in a relatively high current density ( Je ~ 107A/m2) at the surface. Because the ZrO is easily poisoned by ambient gases, the Schottky source requires a vacuum substantially better than that of a LaB6 source.
iii. Field emission
If the electrostatic field at a tip of a cathode is increased sufficiently, the width (horizontal in Fig.3-4) of the potential barrier becomes small enough to allow electrons to escape through the surface potential barrier by quantum-mechanical tunneling, a process known as field emission.
The probability of electron tunneling becomes high when the barrier width, w is comparable to de Broglie wavelength of the electron. This wavelength is related to the electron momentum p by p=h/Î» where h= 6.63 x 10-34 Js is the Planck constant. Because the barrier width is smallest for electrons at the top of the conduction band, they are the ones most likely to escape.
Because thermal excitation is not required, a field-emission tip can operate at room temperature, and the process is sometimes called cold field emission. As there is no evaporation of tungsten during normal operation, the tip can last for many months or even years before replacement. It is heated (“flashed”) from time to time to remove adsorbed gases, which affect the work function and cause the emission current to be unstable. Even so, cold field emission requires ultra-high vacuum (UHV: pressure ~ 10-8 Pa) to achieve stable operation, requiring an elaborate vacuum system and resulting in substantially greater cost of the instrument.
B. Electromagnetic Lens System
The TEM may be required to produce a highly magnified (e.g, M = 105) image of a specimen on a fluorescent screen, of diameter typically 15 cm. To ensure that the screen image is not too dim, most of the electrons that pass through the specimen should fall within this diameter, which is equivalent to a diameter of (15 cm)/M = 1.5 µm at the specimen. For viewing larger areas of specimen, however, the final-image magnification might need to be as low as 2000, requiring an illumination diameter of 75 µm at the specimen. In order to achieve the required flexibility, the condenser-lens system must contain at least two electron lenses.
The first condenser (C1) lens is a strong magnetic lens, with a focal length f that may be as small as 2 mm. Using the virtual electron source(diameter ds) as its object, C1 produces areal image of diameter d1. Because the lens is located 20 cm or more below the object, the object distance, u ~ 20 cm >> f and so the image distance v ~ f.
The second condenser (C2) lens is a weak magnetic lens ( f ~ several centimeters) that provides little or no magnification (M ~ 1) but allows the diameter of illumination (d) at the specimen to be varied continuously over a wide range. The C2 lens also contains the condenser aperture (the hole in the condenser diaphragm) whose diameter D can be changed in order to control the convergence semi-angle of the illumination, the maximum angle by which the incident electrons deviate from the optic axis.
Figure shows lens action within the accelerating field of an electron gun, between the electron source and the anode. Curvature of the equipotential surfaces around the hole in the Wehnelt electrode constitutes a converging electrostatic lens (equivalent to a convex lens in light optics), whereas the non-uniform field just above the aperture in the anode creates a diverging lens (the equivalent of a concave lens in light optics).
C. Sample Holder
To allow observation in different brands or models of microscope, TEM specimens are always made circular with a diameter of 3 mm. Perpendicular to this disk, the specimen must be thin enough (at least in some regions) to allow electrons to be transmitted to form the magnified image. The specimen stage is designed to hold the specimen as stationary as possible, as any drift or vibration would be magnified in the final image, impairing its spatial resolution (especially if the image is recorded by a camera over a period of several seconds). But in order to view all possible regions of the specimen, it is also necessary to move the specimen horizontally over a distance of up to3 mm if necessary.
The design of the stage must also allow the specimen to be inserted into the vacuum of the TEM column without introducing air. This is achieved by inserting the specimen through an airlock, a small chamber into which the specimen is placed initially and which can be evacuated before the specimen enters the TEM column. Not surprisingly, the specimen stage and airlock are the most mechanically complex and precision-machined parts of the TEM. There are two basic designs of the specimen stage: side-entry and top-entry.
In a side-entry stage, the specimen is clamped (for example, by a threaded ring) close to the end of a rod-shaped specimen holder and is inserted horizontally through the airlock. The airlock-evacuation valve and a high-vacuum valve (at the entrance to the TEM column) are activated by rotation of the specimen holder about its long axis; see figure (a).
One advantage of this side-entry design is that it is easy to arrange for precision motion of the specimen. Translation in the horizontal plane (x and y directions) and in the vertical (z) direction is often achieved by applying the appropriate movement to an end-stop that makes contact with the pointed end of the specimen holder. A further advantage of the side-entry stage is that heating of a specimen is easy to arrange, by installing a small heater at the end of the specimen holder, with electrical leads running along the inside of the holder to a power supply located outside the TEM. The ability to change the temperature of a specimen allows structural changes in a material (such as phase transitions)to be studied at the microscopic level. Specimen cooling can also be achieved, by incorporating (inside the side-entry holder) a heat-conducting metal rod whose outer end is immersed in liquid nitrogen (at 77 K).
One disadvantage of the side-entry design is that mechanical vibration picked up from the TEM column or from acoustical vibrations in the external air, is transmitted directly to the specimen. In addition, any thermal expansion of the specimen holder can cause drift of the specimen and of the TEM image. These problems have been largely overcome by careful design, including choice of materials used to construct the specimen holder. As a result, side-entry holders are widely used, even for high-resolution imaging.
In a top-entry stage, the specimen is clamped to the bottom end of a cylindrical holder that is equipped with a conical collar; see Figure (b). The holder is loaded into position through an airlock by means of a sliding and tilting arm, which is then detached and retracted. Inside the TEM, the cone of the specimen holder fits snugly into a conical well of the specimen stage, which can be translated in the (x and y) horizontal directions by a precision gear mechanism. The major advantage of a top-entry design is that the loading arm is disengaged after the specimen is loaded, so the specimen holder is less liable to pick up vibrations from the TEM environment. In addition, its axially symmetric design tends to ensure that any thermal expansion occurs radially about the optic axis and therefore becomes small close to the axis.
However, in disadvantage views, it is more difficult to provide tilting, heating, or cooling of the specimen. Although such facilities have all been implemented in top-entry stages, they require elaborate precision engineering, making the holder fragile and expensive. Because the specimen is held at the bottom of its holder, it is difficult to collect more than a small fraction of the x-rays that are generated by the transmitted beam and emitted in the upward direction, making this design less attractive for high-sensitivity elemental analysis.
D. Imaging System
The sample is placed in front of the objective lens in a form of thin foil, thin section or fine particles transparent for the electron beam. (Figure. 3). The objective lens forms an image of the electron density distribution at the exit surface of the specimen based on the electron optical principles. The diffraction, projection and intermediate lenses below the objective lens are used to focus and magnify either the diffraction pattern or the image onto a fluorescent screen, which converts the electrons into visible light signal. There are three important mechanisms, which produce image contrast in the electron microscope: mass-thickness contrast, phase contrast and diffraction or amplitude contrast.
i. Mass-thickness contrast arises from incoherent elastic scattering of electrons. As electrons go through the specimen they are scattered off axis by elastic nuclear interaction also called Rutherford scattering. The cross section for elastic scattering is a function of the atomic number (Z). As the thickness of the specimen increases the elastic scattering also increases since the mean-free path remains fixed.
Also specimens consisting of higher Z elements will scatter more electrons than low-Z specimens. This will create differential intensity in an image formed from thicker regions where fewer electrons will be transmitted to the image compared to a thinner or low atomic number region, which will be brighter in the image plane. In TEM, the mass-thickness contrast is affected by the size of the objective aperture and the accelerating voltage. Smaller apertures will increase the difference in the ratio of scattered and transmitted electrons and as a consequence will increase the contrast between regions of different thickness of mass. Lowering the accelerating voltage will lead to similar effect since the scattering angle and the cross section increase which also will cause increase in the relative contrast between higher mass and lower mass regions.
ii. Phase contrast. Some of the electrons leaving the specimen are recombined to form the image so that phase differences present at the exit surface of the specimen are converted into intensity differences in the image. Phase contrast is the dominant mechanism for object detail <10 Å and is important in lattice resolution studies and investigations of the early stages of short-range order and amorphous materials.
iii. Diffraction contrast. Diffracted electrons leaving the lower surface of a crystalline specimen are intercepted by the objective aperture and prevented from contributing to the image. Alternatively only one diffracted beam forms the image. Diffraction contrast is the dominant mechanism delineating object detail >15 Å in crystalline specimens and is important and widely used contrast mechanism for study of crystal defects. Using this approach considerable quantitative information about the defect structure of the specimen may be obtained without operating the microscope at maximum resolution.
Electron microscopes cannot operate in air for a number of reasons. The penetration of electrons through air is typically no more than 1 meter, so after coming on meter from the gun, the whole beam would be lost to collisions of the electrons with the air molecules. It is also not possible to generate the high charge difference between the anode and cathode in the gun because air is not a perfect insulator. Finally, the beam on the specimen while in air would trap all sorts of rubbish (air is full of hydrocarbon molecules) on the specimen, crack them (removing hydrogen, oxygen, etc.) and thus leave a thick carbon contamination layer on the specimen. Each electron microscope therefore has a vacuum system. The degree of sophistication of the vacuum system depends on the requirements. Simple imaging of biological thin sections is much less demanding than cryo applications or small-probe analysis in materials science and a thermionic gun can operate under much worse vacuum than a Field Emission Gun (FEG).
The most basic vacuum system consists of a vessel connected to a pump that removes the air. The vacuum system of an electron microscope is considerably more complicated, containing a number of vessels, pumps, valves (to separate different vessels) and gauges (to measure vacuum pressures). From the bottom up we can distinguish four vessels in the vacuum system:
The buffer tank
The projection chamber
The column (specimen area)
The electron gun area
Sometimes a tubomolecular pump (TMP), essentially a high-speed turbine fan, is used in place of (or to supplement) a diffusion pump. Usually an ion pump is used to achieve pressures below 10-4Pa, as required to operate a LaB6, Schottky, or field-emission electron source. By applying a potential difference of several kilovolts between large electrodes, a low-pressure discharge is set up (aided by the presence of a magnetic field) which removes gas molecules by burying them in one of the electrodes.
Figure shows cross section through a diffusion pump. The arrows show oil vapor leaving jets within the baffle assembly. Water flowing within a coiled metal tube keeps the walls cool.
Frequently, liquid nitrogen is used to help in achieving adequate vacuum inside the TEM, through a process known as cryo
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