Electron microscopy

Editor's Notes

1. From the dawn of science there has been an interest in being able to look at smaller and smaller details of the world around us. Biologists have wanted toexamine the structure of cells, bacteria, viruses, and colloidal particles. Materials scientists have wanted to see inhomogeneities and imperfections in metals, crystals, and ceramics.In geology, the detailed study of rocks, minerals, and fossils on a microscopic scale provides insight into the origins of our planet and its valuable mineral resources.
2. For example, try looking at a newspaper picture, or one in a magazine, through a magnifying glass. You will see that the image,is actually made up of dots too small and too close together to be,separately resolved by your eye alone. The same phenomenon will be observed on an LCD computer display or flat screen TV when magnified to reveal the individual “pixels” that make up the image
3. Optical microscopes are the ones most familiar to everyone. They use visible light and transparent lenses to see objects as small as about one micrometer (one millionth of a meter), such as a red blood cell (7 μm) or a human hair (100 μm).
4. With visible light it was impossible to resolve points in the object that were closer together than a few hundred nanometers
5. similar to the way glass lenses are used to bend and focus visible light.
6. In the 1920s, it was discovered that accelerated electrons behave in vacuum much like light. They travel in straight lines and have wavelike properties, with a wavelength that is about 100,000 times shorter than that of visible light. Furthermore, it was found that electric and magnetic fields could be used to shape the paths followed by electrons similar to the way glass lenses are used to bend and focus visible light. Ernst Ruska at the University of Berlin combined these characteristics and built the first transmission electron microscope (TEM) in 1931. For this and subsequent work on the subject, he was awarded the Nobel Prize for Physics in 1986. The first electron microscope used two magnetic lenses, and three years later he added a third lens and demonstrated a resolution of 100 nm, twice as good as that of the light microscope. Today, electron microscopes have reached resolutions of better than 0.05 nm, more than 4000 times better than a typical light microscope and 4,000,000 times better than the unaided eye.
7. The positively charged protons and neutral neutrons are held tightly together in a central nucleus. Negatively charged electrons surround the nucleus.Normally, the number of protons equals the number of electrons so that the atom as a whole is neutral. When an atom deviates from this normal configuration by losing or gaining electrons, it acquires a net positive or negative charge and is referred to as an ion.
8. The transmission electron microscope (TEM) was the first type of Electron Microscope to be developedPasses a beam of electrons through the specimen.The electrons detected on a fluorescent screen on which the image is displayed.Thin sections of specimen -as electrons have to pass through the specimen for the image to be produced.This is the most common form of electron microscope and has the best resolution [0.2nm] .
9. The electron beam from the electron gun can be focussed and defocussed by a series of electro- magnetic lenses. Similar to the light microscope, the "Condenser Lenses" concentrate the beam onto the specimen. Electrons passing through the specimen will be focussed by the "Objective" &"Intermediate" lenses to form an intermediate image.The "Projector lens" enlarges this image into a final image on the fluorescent viewing screen at the bottom of the microscope column.Each lens is basically a circular electro-magnet. A variable electric current through the lens will produce a magnetic field of variable strengths which will deflect or bend the electron beam passing through
10. caused by collisions between the beam electrons and the atoms of the specimen
11. Specimen interaction is what makes Electron Microscopy possible. The energetic electrons in the microscope strike the sample and various reactions can occur as shown below. The reactions noted on the top side of the diagram are utilized when examining thick or bulk specimens (SEM) while the reactions on the bottom side are those examined in thin or foil specimens (TEM).
12. The incident electron is then scattered "backward 180 degrees- backscaterred electron.Sec electron- Caused by an incident electron passing "near" an atom in the specimen, near enough to impart some of its energy to a lower energy electron (usually in the K-shell). This causes a slight energy loss and path change in the incident electron and the ionization of the electron in the specimen atom. This ionized electron then leaves the atom with a very small kinetic energy (5eV) and is then termed a "secondary electron". Each incident electron can produce several secondary electron.Auger Electrons-Caused by the de-energization of the specimen atom after a secondary electron is produced. Since a lower (usually K-shell) electron was emitted from the atom during the secondary electron process an inner (lower energy) shell now has a vacancy. A higher energy electron from the same atom can "fall" to a lower energy, filling the vacancy. This creates and energy surplus in the atom which can be corrected by emitting an outer (lower energy) electron; an Auger Electron.
13. Mass absorption contrastOn passing through matter, a beam of electrons is gradually attenuated. The degree of attenuation increases with the thickness of the specimen and its mass, so variations of mass and thickness across the sample give rise to contrast in the image.Diffraction contrastDiffraction of electrons from Bragg planes causes a change in their direction of travel. Hence, contrast can arise between adjacent grains or between different regions near the core of a dislocation.Phase contrastScattering mechanisms often cause a change in the phase of the scattered electrons, as well as a change in direction. Interference between electrons of different phase which are incident on the same part of the image will cause a change in intensity and give rise to contrast.
14. Biological material (light elements):Only few electrons escape from specimenAlmost no contrast, similar contrast everywhere on specimenBlurred image (electrons from “large” volume)Contrast enhancement important & needed:Localization of the signal to the surfaceCoating of biological specimen with thin heavy metal layer (a few nm)Reducing acceleration voltage
15. Electron microphotograph showing myelin figure (´ 10,000) and Zebra body-inset (´ 15,000) (Uranyl acetate and lead citrate stain).
16. he typical ciliary axoneme consists of 2 central microtubules surrounded by 9 microtubular doublets. Each doublet has an A subunit and a B subunit attached as a semicircle. A central sheath envelops the 2 central microtubules, which attach to the outer doublets by radial spokes.The outer doublets are interconnected by nexin links, and each A subunit is attached to 2 dynein arms that contain adenosine triphosphatase; one inner arm and one outer arm. The primary function of the central sheath, radial spokes, and nexin links is to maintain the structural integrity of the cilium, whereas the dynein arms are responsible for ciliary motion.
17. Mitochondrial myopathy, ultrastructure. A large number of enormous mitochondria can be seen in the intermyofibrillar network of this myofiber. These mitochondria are larger than entire sarcomeres. Normal mitochondria are much smaller than sarcomeres.
18. The four stages of melanosome development are shown in the upper panels. Note the dense bilayered coat (arrowhead) and intraluminal vesicles (arrow) of stage I melanosomes, the proteinaceous fibrils (arrow) of stage II, and the melanin deposition (black) in stages III and IV.