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- 2. Electron Microscopy Techniques Introduction •Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. • The main advantage of Electron Microscopy is the unusual short wavelength of the electron beams, substituted for light energy. • The wavelengths of about 0.005 nm increases the resolving power of the instrument to fractions
- 3. Microscopy • Main branches:optical, electron and scanning probe microscopy. • Optical and electron microscopy involves the diffraction, reflection, or refraction of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. • Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest.
- 4. Optical microscopy -definition • Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. • The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. • The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope.
- 5. Optical microscopy -scheme
- 6. Optical microscopy -magnification
- 7. Electron Microscopy • Beamsof electrons are used to produce images • Wavelength of electron beam is much shorter than light, resulting in much higher resolution
- 8. Types of ElectronMicroscope • Transmission Electron Microscope (TEM) uses a wide beam of electrons passing through a thin sliced specimen to form an image. This microscope is analogous to a standard upright or inverted light microscope • Scanning Electron Microscope (SEM) uses focused beam of electrons scanning over the surface of thick or thin specimens. Images are produced one spot at a time in a grid-like raster pattern.
- 9. Introduction • Transmission electronmicroscopy (TEM, also sometimes conventional transmission electron microscopy or CTEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. • The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. • An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. • The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled device.
- 10. • Transmission electronmicroscopes are capable of imaging at a significantly higher resolution than light microscopes, owing to the smaller de Broglie wavelength of electrons. • This enables the instrument to capture fine detail—even as small as a single column of atoms, which is thousands of times smaller than a resolvable object seen in a light microscope. • Transmission electron microscopy is a major analytical method in the physical, chemical and biological sciences. • TEMs find application in cancer research, virology, and materials science as well as polluton, nanotechnology and semiconductor research.
- 12. Transmission Electron Microscopy(TEM) • Ultrathin sections of specimens • Light passes through specimen, then an electromagnetic lens, to a screen or film • Specimens may be stained with heavy metal salts
- 13. Transmission Electron Microscopy(TEM) • 10,000–100,000×; resolution 2.5 nm
- 14. The principles ofTEM • Transmission electron microscopy uses high energy electrons (up to 300 kV accelerating voltage) which are accelerated to nearly the speed of light. • The electron beam behaves like a wavefront with wavelength about a million times shorter than lightwaves. • When an electron beam passes through a thin-section specimen of a material, electrons are scattered. • A sophisticated system of electromagnetic lenses focuses the scattered electrons into an image or a diffraction pattern, or a nano-analytical spectrum, depending on the mode of operation.
- 15. • Each ofthese modes offers a different insight about the specimen. The imaging mode provides a highly magnified view of the micro- and nanostructure and ultimately, in the high resolution imaging mode a direct map of atomic arrangements can be obtained (high resolution EM = HREM). • The diffraction mode (electron diffraction) displays accurate information about the local crystal structure. The nanoanalytical modes (x-ray and electron spectrometry) tell researchers which elements are present in the tiny volume of material. • These modes of operation provide valuable information for scientists and engineers in search of stronger materials, faster microchips, or smaller nanocrystals.
- 16. Parts of themachine • The typical transmission electron microscope laboratory contains a machine with these components: I. Electron gun II. Electron column III. Electro-magnetic lens system IV. Detectors V. Water chilling system VI. Specimen/sample chamber VII. Main control panel and operational controls VIII. Image capture
- 18. Electron gun • Theelectron gun generates the electron beam. It is usually positioned in the top of the instrument column. • The emitter is seated within a cone-shaped Wehnelt cylinder and the beam travels out of the small central hole shown in the apex of the cone.
- 19. Electron column • Theelectron column is made up of the gun assembly at the top, a column filled with a set of electromagnetic lenses, the sample port and airlock, and a set of apertures that can be moved in and out of the path of the beam. The contents of the column are under vacuum.
- 20. Electron lens • Electronlenses are designed to act in a manner emulating that of an optical lens, by focusing parallel electrons at some constant focal distance. • Electron lenses may operate electrostatically or magnetically. The majority of electron lenses for TEM use electromagnetic coils to generate a convex lens. • The field produced for the lens must be radially symmetrical, as deviation from the radial symmetry of the magnetic lens causes aberrations such as astigmatism, and worsens spherical and chromatic aberration. • Electron lenses are manufactured from iron, iron-cobalt or nickel cobalt alloys, such as permalloy.
- 21. These are selectedfor their magnetic properties, such as magnetic saturation, hysteresis and permeability.
- 22. Detectors • One ofthe most common detectors seen on a transmission electron microscope is the x-ray energy dispersive spectroscopy (EDS or EDX) system. This typically involves a large dewar for liquid nitrogen (to keep the detector cold), an arm on which the equipment sits, and a solid state detector that penetrates the column (arrow) so it is located near the sample.
- 23. Apertures • Apertures areannular 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.
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- 25. A ray diagramfor the diffraction mechanism in TEM
- 27. Transmission Electron Microscope(TEM) Working Concept • TEM works much like a slide projector. • A projector shines a beam of light through (transmits) the slide, as the light passes through it is affected by the structures and objects on the slide. • These effects result in only certain parts of the light beam being transmitted through certain parts of the slide. • This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide. • TEMs work the same way except that they shine a beam of electrons (like the light) through the specimen (like the slide). • Whatever part is transmitted is projected onto a phosphor screen for the user to see. • A more technical explanation of typical TEMs workings is as follows.
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- 29. • The "VirtualSource" 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)
- 30. • The beamstrikes the specimen and parts of it are transmitted. • This transmitted portion is focused by the objective lens into an image • The image is passed down the column through the 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
- 31. Comparison b/w SEMand TEM
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- 39. Image Modes • InTEM, absorption of electrons plays a very minor role in image formation. TEM contrast relies instead on deflection of electrons from their primary transmission direction when they pass through the specimen. The contrast is generated when there is a difference in the number of electrons being scattered away from the transmitted beam. There are two mechanisms by which electron scattering creates images: mass-density contrast and diffraction contrast.
- 40. Mass-Density Contrast • Thedeflection of electron scan result from interaction between electrons and an atomic nucleus. Deflection of an electron by an atomic nucleus, which has much more mass than an electron, is like a collision between a particle and a wall. The particle (electron) changes its path after collision. The amount of electron scattering at any specific point in a specimen depends on the mass-density (product of density and thickness) at that point. Thus, difference in thickness and density in a specimen will generate variation in electron intensity received by an image screen in the TEM.
- 41. • The deflectedelectron with scattering angle larger than the α angle (in the order of 0.01 radians) controlled by the objective aperture will be blocked by the aperture ring. Thus, the aperture reduces the intensity of the transmission beam as the beam passes through it. The brightness of the image is determined by the intensity of the electron beam leaving the lower surface of the specimen and passing through the objective aperture. The intensity of the transmitted beam (It) is the intensity of primary beam (Io) less the intensity of beam deflected by object (Id) in a specimen. Specimen.
- 43. Diffraction Contrast • Wecan also generate contrast in the TEM by a diffraction method. • Diffraction contrast is the primary mechanism of TEM image formation in crystalline specimens. Diffraction can be regarded as collective deflection of electrons. • Electrons can be scattered collaboratively by parallel crystal planes similar to X-rays. Bragg’s Law, which applies to X-ray diffraction, also applies to electron diffraction. • When the Bragg conditions are satisfied at certain angles between electron beams and crystal orientation, constructive diffraction occurs and strong electron deflection in a specimen results. • Thus, the intensity of the transmitted beam is reduced when the objective aperture blocks the diffraction beams, similar to the situation of mass-density contrast.
- 45. • Note thatthe main difference between the two contrasts is that the diffraction contrast is very sensitive to specimen tilting in the specimen holder but mass-density contrast is only sensitive to total mass in thickness per surface area. • The diffraction angle (2θ) in a TEM is very small (≤1◦) and the diffracted beam from a crystallographic plane (hkl) can be focused as a single spot on the back-focal plane of the objective lens. The Ewald sphere is particularly useful for interpreting electron diffraction in the TEM. • When the transmitted beam is parallel to a crystallographic axis, all the diffraction points from the same crystal zone will form a diffraction pattern (a reciprocal lattice) on the back-focal plane. The diffraction contrast can generate bright-field and dark-field TEM images. • In order to understand the formation of bright-field and dark-field images, the diffraction mode in a TEM must be mentioned. A TEM can be operated in two modes : the image mode and the diffraction mode.
- 47. Phase Contrast • Bothmass-density and diffraction contrasts are amplitude contrast because they use only the amplitude change of transmitted electron waves. • A TEM can also use phase difference in electron waves to generate contrast. The phase contrast mechanism, however, is much more complicated than that of light microscopy. T • he TEM phase contrast produces the highest resolution of lattice and structure images for crystalline materials. • Thus, phase contrast is often referred as to high resolution transmission electron microscopy (HRTEM). • Phase contrast must involve at least two electron waves that are different in wave phase. • Thus, we should allow at least two beams (the transmitted beam and a diffraction beam) to participate in image formation in a TEM.
- 48. • A crystallinespecimen with a periodic lattice structure generates a phase difference between the transmitted and diffracted beams. • The objective lens generates additional phase difference among the beams. • Recombination of transmitted and diffracted beams will generate an interference pattern with periodic dark–bright changes on the image plane because of beam interferences. • An interference pattern is a fringe type that reveals the periodic nature of a crystal. • Theoretical interpretation of phase contrast images is complicated, particularly when more than one diffraction spot participates in image formation.
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- 52. TEM Imaging • ATransmission Electron Microscope produces a high-resolution, black and white image from the interaction that takes place between prepared samples and energetic electrons in the vacuum chamber. • Air needs to be pumped out of the vacuum chamber, creating a space where electrons are able to move. • The electrons then pass through multiple electromagnetic lenses. These solenoids are tubes with coil wrapped around them. • The beam passes through the solenoids, down the column, makes contact with the screen where the electrons are converted to light and form an image. • The image can be manipulated by adjusting the voltage of the gun to accelerate or decrease the speed of electrons as well as changing the electromagnetic wavelength via the solenoids. • The coils focus images onto a screen or photographic plate.
- 53. • During transmission,the speed of electrons directly correlates to electron wavelength; the faster electrons move, the shorter wavelength and the greater the quality and detail of the image. • The lighter areas of the image represent the places where a greater number of electrons were able to pass through the sample and the darker areas reflect the dense areas of the object. • These differences provide information on the structure, texture, shape and size of the sample. • To obtain a TEM analysis, samples need to have certain properties. They need to be sliced thin enough for electrons to pass through, a property known as electron transparency. • Samples need to be able to withstand the vacuum chamber and often require special preparation before viewing. • Types of preparation include dehydration, sputter coating of non- conductive materials, cryofixation, sectioning and staining.
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- 55. Advantages of TEM •A Transmission Electron Microscope is an impressive instrument with a number of advantages such as: • TEMs offer the most powerful magnification, potentially over one million times or more • TEMs have a wide-range of applications and can be utilized in a variety of different scientific, educational and industrial fields • TEMs provide information on element and compound structure • Images are high-quality and detailed • TEMs are able to yield information of surface features, shape, size and structure • They are easy to operate with proper training
- 56. Disadvantages of TEM •TEMs are large and very expensive • Laborious sample preparation • Potential artifacts from sample preparation • Operation and analysis requires special training • Samples are limited to those that are electron transparent, able to tolerate the vacuum chamber and small enough to fit in the chamber • TEMs require special housing and maintenance • Images are black and white Electron microscopes are sensitive to vibration and electromagnetic fields and must be housed in an area that isolates them from possible exposure. A Transmission Electron Microscope requires constant upkeep including maintaining voltage, currents to the electromagnetic coils and cooling water.