Both optical microscope and electron microscope
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Saturday, November 1, 2008
Wednesday, October 29, 2008
Microscope
Electron microscope
Accelerated electrons behave in vacuum just like light. They travel in straight lines and have wavelength which is about 100, 000 times smaller than that of light.
Electric and magnetic fields have the same effect on electron as glass lenses and mirrors have on visible light.
Transmission electron microscope (TEM)
Transmission electron microscope can be compared with slide projector. In TEM, light source is replaced by an electron source tungsten filament heated in vacuum. Glass lenses are replaced by electromagnetic lenses and projection screen is replaced by fluorescent screen which emits light when struck by electrons. The whole trajectory from source of electron to screen is under vacuum and the specimen has to be very thin to allow the electrons to penetrate it. Electron are easily stopped or deflected by air molecules. That is why the microscope has to be evacuated and why specimens for TEM have to be very thin in order to imaged with electrons.
Unlike glass lenses, electromagnetic lenses are variable. By varying the current through the lens coil, the focal length, which determines the magnification, can be varied. In light microscope, variation in magnification is obtained by changing lens or by mechanically moving lens.
Electron gun comprises s filament and an anode. The tungsten filament is hairpin shaped and heated to about 2700 OC. By applying a very high potential difference between filament and anode, electrons are extracted and accelerated towards anode. Anode has a hole through which electron beam emerges. The beam is condensed by condenser coarse and fine condenser, then focused on specimen through condenser aperture that knocks out high angle electrons far from optic axis.
When electrons impinge on specimen, a number of things can happen
Some of the electrons are absorbed
Some are scattered over small angles
In crystalline specimens, electrons are scattered in very distinct directions
Some of impinging electrons are reflected (backscattered electrons)
Impinging electrons cause the specimen to emit electrons (secondary electron)
Impinging electrons cause the specimen to emit X rays
Impinging electrons cause the specimen to emit photons or light (cathodoluminescence)
Transmitted
Transmitted portion is focused by objective lens into an image. Objective aperture and selected area aperture enhance contrast by blocking high angle diffracted electrons. Image is passed through intermediate and projector lens being enlarged all the way. The image strikes the phosphor image screen and light is generated allowing user to see the image. Darker areas of image represent the area on sample that transmitted fewer electrons (thicker or dense). Light areas of image represent those areas of sample through which more electrons were transmitted (thinner).
Specimen preparation
In biology, tissues are treated as follows
First, there is a chemical treatment to remove water and preserve the tissue as much as possible in its original state.
It is then embedded in a hardening resin
After the resin has hardened, slices with an average thickness of 0.5 µm are cut with an instrument called ultramicrotome equipped with glass or diamond knife.
Tiny sections thus obtained are placed on specimen carrier coated with a structureless carbon film
Scanning electron microscope (SEM)
The electron gun produces an electron beam which is focused into a fine spot less than 4 nm in diameter on the specimen. The stream of electron is coarsely condensed by first condenser lens. It works in conjunction with condenser aperture to eliminate high angle electrons from the beam. Second condenser lens focus electron into finely thin beam. Objective aperture further eliminates high angle electron from the beam. The beam is scanned over the specimen in a series of lines and frames called raster (like in CRT) by set of coils. The final lens, objective focuses the scanning beam onto the very small area of the specimen. Several things may happen to these electrons. Commonly image formation is by means of low energy secondary electrons. An image is built up simply by scanning the electron beam across the specimen in exact synchrony with the scan of the electron beam in CRT. Magnification results from the ratio of the area scanned on the specimen to the area of television screen. Therefore, magnification is increased in SEM by scanning electron beam over a smaller area of the specimen. Detectors for secondary electron are usually scintillation detector or solid state detector.
Specimen preparation
Specimens must be able to withstand the vacuum of the chamber and the electron bombardment. Many specimens can be brought into the chamber without preparation of any kind. If the specimen contains any volatile components such as water, this should be removed using a drying process or in some circumstances it can be frozen to solid. Specimens must be electrically conductive, at least at the surface. Non conducting specimens will charge up under electron bombardment and need to be coated with a conducting layer. Since heavy element like gold produces good secondary electron and also yields good quality image, this is favorable element for coating. All in all, preparation of specimen to be investigated by SEM is not as complicated as the preparation of specimen for TEM.
Disadvantages of electron microscope
Expensive to build and maintain
Require extremely stable high voltage supplies, extremely stable current to each electromagnetic coils/lens
Continuously pumped high or ultra high vacuum required
As they are sensitive to vibration and external magnetic field, EM should be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling system
TEM has the best resolution
Accelerated electrons behave in vacuum just like light. They travel in straight lines and have wavelength which is about 100, 000 times smaller than that of light.
Electric and magnetic fields have the same effect on electron as glass lenses and mirrors have on visible light.
Transmission electron microscope (TEM)
Transmission electron microscope can be compared with slide projector. In TEM, light source is replaced by an electron source tungsten filament heated in vacuum. Glass lenses are replaced by electromagnetic lenses and projection screen is replaced by fluorescent screen which emits light when struck by electrons. The whole trajectory from source of electron to screen is under vacuum and the specimen has to be very thin to allow the electrons to penetrate it. Electron are easily stopped or deflected by air molecules. That is why the microscope has to be evacuated and why specimens for TEM have to be very thin in order to imaged with electrons.
Unlike glass lenses, electromagnetic lenses are variable. By varying the current through the lens coil, the focal length, which determines the magnification, can be varied. In light microscope, variation in magnification is obtained by changing lens or by mechanically moving lens.
Electron gun comprises s filament and an anode. The tungsten filament is hairpin shaped and heated to about 2700 OC. By applying a very high potential difference between filament and anode, electrons are extracted and accelerated towards anode. Anode has a hole through which electron beam emerges. The beam is condensed by condenser coarse and fine condenser, then focused on specimen through condenser aperture that knocks out high angle electrons far from optic axis.
When electrons impinge on specimen, a number of things can happen
Some of the electrons are absorbed
Some are scattered over small angles
In crystalline specimens, electrons are scattered in very distinct directions
Some of impinging electrons are reflected (backscattered electrons)
Impinging electrons cause the specimen to emit electrons (secondary electron)
Impinging electrons cause the specimen to emit X rays
Impinging electrons cause the specimen to emit photons or light (cathodoluminescence)
Transmitted
Transmitted portion is focused by objective lens into an image. Objective aperture and selected area aperture enhance contrast by blocking high angle diffracted electrons. Image is passed through intermediate and projector lens being enlarged all the way. The image strikes the phosphor image screen and light is generated allowing user to see the image. Darker areas of image represent the area on sample that transmitted fewer electrons (thicker or dense). Light areas of image represent those areas of sample through which more electrons were transmitted (thinner).
Specimen preparation
In biology, tissues are treated as follows
First, there is a chemical treatment to remove water and preserve the tissue as much as possible in its original state.
It is then embedded in a hardening resin
After the resin has hardened, slices with an average thickness of 0.5 µm are cut with an instrument called ultramicrotome equipped with glass or diamond knife.
Tiny sections thus obtained are placed on specimen carrier coated with a structureless carbon film
Scanning electron microscope (SEM)
The electron gun produces an electron beam which is focused into a fine spot less than 4 nm in diameter on the specimen. The stream of electron is coarsely condensed by first condenser lens. It works in conjunction with condenser aperture to eliminate high angle electrons from the beam. Second condenser lens focus electron into finely thin beam. Objective aperture further eliminates high angle electron from the beam. The beam is scanned over the specimen in a series of lines and frames called raster (like in CRT) by set of coils. The final lens, objective focuses the scanning beam onto the very small area of the specimen. Several things may happen to these electrons. Commonly image formation is by means of low energy secondary electrons. An image is built up simply by scanning the electron beam across the specimen in exact synchrony with the scan of the electron beam in CRT. Magnification results from the ratio of the area scanned on the specimen to the area of television screen. Therefore, magnification is increased in SEM by scanning electron beam over a smaller area of the specimen. Detectors for secondary electron are usually scintillation detector or solid state detector.
Specimen preparation
Specimens must be able to withstand the vacuum of the chamber and the electron bombardment. Many specimens can be brought into the chamber without preparation of any kind. If the specimen contains any volatile components such as water, this should be removed using a drying process or in some circumstances it can be frozen to solid. Specimens must be electrically conductive, at least at the surface. Non conducting specimens will charge up under electron bombardment and need to be coated with a conducting layer. Since heavy element like gold produces good secondary electron and also yields good quality image, this is favorable element for coating. All in all, preparation of specimen to be investigated by SEM is not as complicated as the preparation of specimen for TEM.
Disadvantages of electron microscope
Expensive to build and maintain
Require extremely stable high voltage supplies, extremely stable current to each electromagnetic coils/lens
Continuously pumped high or ultra high vacuum required
As they are sensitive to vibration and external magnetic field, EM should be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling system
TEM has the best resolution
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