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
Wednesday, October 29, 2008
Monday, October 20, 2008
Microscope
Light microscope
Microscope is the instrument most characteristics of the microbiological laboratory. The magnification it provides enables us to see microorganisms and their structures otherwise invisible to the naked eye. The magnification attainable by microscope ranges from X100 to X400, 000. Several different kinds of microscopy are available many techniques have been developed by which specimens can be prepared for examination. Each type of microscopy and each method of preparing specimen offers advantages for demonstration of specific morphological features.
There are two fundamentally different types of microscope. Light microscope and electron microscope. Light microscope uses a series of glass lenses to focus light in order to form an image. Maximum magnification attained is X1, 500. Electron microscope uses electromagnetic lenses to focus a beam of electrons. Maximum magnification attained is X400, 000.
Both living and dead specimens can be viewed with light microscope and often in real color.
Only dead microorganisms are viewed with electron microscope and never in real color.
Magnification is not the best measure of a microscope. Resolution or resolving power, the ability to distinguish two adjacent points as distinct and separate in a specimen, is much more reliable estimate of a microscope’s utility. Greater magnification without greater resolution i.e., mere increase in size without the ability to distinguish structural details is not beneficial. The largest magnification produced by a microscope may not be the most useful because the image obtained may be unclear or fuzzy. Magnification beyond the resolving power is of no value and is called empty or useless magnification. The resolving power of a microscope is a function of the wavelength of light used and the numerical aperture of the lens system. Total magnification of the system is determined by multiplying the magnifying power of the objective by that of eyepiece.
Resolution of light microscope is 0.5 µm approximately.
Resolution of electron microscope is upto 1 nm
In bright field microscopy, the microscopic field is brightly lighted and microorganisms appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light but staining them with dye greatly increases their light absorbing ability resulting in greater contrast and color differentiation.
All modern light microscopes are made up of more than one glass lens in combination. The major components are condenser lens, the objective lens and the eyepiece lens. Each of these components is in turn made up of combinations of lenses which are necessary to produce magnified images.
There are two basic types of compound light microscope stand- upright and inverted microscope. Upright microscope is for viewing specimens. Condenser lens and light source are below specimen. Stage is movable.
Inverted microscope is for manipulation of specimen directly on stage. E.g,. microinjection of macromolecules into tissue culture cells, invitro fertilization of eggs etc. condenser lens and source of light are above specimen. Objective is movable.
Numerical aperture is a measure of the ability of lens to collect light from the specimen. Lenses with low numerical aperture collect less light than those with higher numerical aperture. Higher numerical aperture objective yields best resolution. The angle theta subtended by optical axis and the outermost rays still covered by objective lens is the measure of aperture of objective. It is half aperture angle.
The magnitude of this angle is expressed as a sine value.NA=n X sin theta
Sin theta=sine value of half aperture angle
N=refractive index of the medium filling the space between front lens and coverslip.
For dry objective n=1=refractive index of air
For oil immersion n=1.56=refractive index of oil immersion which is equal to refractive index of glass.
The wavelength of light used in optical microscope is also limited. The shorter the wavelength of illuminating light, the higher is the resolving power of the microscope. Visible wave length of light rages between 400 nm and 750 nm. By using ultraviolet light as light source, the resolution can be improved.
Greatest resolution using visible light=200 nm
Greatest resolution using ultraviolet light=100 nm
Greatest resolution in light microscopy is obtained using shortest wavelength of visible light and objective with maximum numerical aperture.
Dark field microscopy
It produces image of brightly illuminated objects on a black background. Brilliant illumination of objects is accomplished by equipping light microscope with a special kind of condenser that transports a hollow cone of light from the source of illumination. Most of the light directed through the condenser does not enter the object. Therefore, field is dark. However, some of the light rays will be diffracted (scattered) if transparent medium contains objects such as microbial cells. This diffracted light will enter the objective and reach the eye. Thus objects or microbial cells appear bright. It is used for viewing motility and outlines of objects in liquid media such as living spermatozoa, microorganisms, cells growing in tissue culture.
Spirochaetes Treponema pallidum causative agent of syphilis STD can’t be seen in Gram stained smears, Borrelia, Leptospira, Vibrio.
Fluorescence microscopy
In fluorescence microscopy, ultraviolet light which has very short wavelength and is not visible to the eye, is used to illuminate organisms, cells, particles which have been previously stained with fluorescing dyes called fluorochromes. These dyes are able to transform the invisible short wavelength ultraviolet light into longer wavelength visible light. The fluorescent stained particles appear glowing against a dark background.
Two types of fluorescence microscopy are used in medical laboratory work- transmitted light fluorescence (TLF) and incident light fluorescence (ILF) also called epifluorescence.
Transmitted light fluorescence –Short wavelength light from a fluorescence lamp such as mercury vapor or quartz halogen lamp passes through primary or excitation filter which removes all unwanted color or wavelength of light and passes only those that are required. The transmission of this filter must match the emission peak of the fluorochromes being used. The light is then brought to the specimen by a dark field immersion condenser. The fluorescence which is given off by the specimen passes through a secondary or barrier filter located between the objective and the eye which filters of all the light other than fluorescence wavelength specific to the specimen
Incident light fluorescence
This involves illuminating the specimen fro above (epifluorescence) using a dichroic mirror which reflects selectively the shorter wavelength radiation and transmits the longer wavelength fluorescence i.e., it is transparent to wavelength above given value and opaque to those below.
The light passes through an excitation filter and is directed onto dichroic mirror located above specimen. The mirror reflects short wavelength excitation light on specimen. The visible light from the reflected specimen passes back through the objective to the dichroic mirror which transmits the longer fluorescent wavelength. A barrier filter ensures that only the fluorescence wavelength specific for the specimen reach the eyepiece. No condenser needed for incident light fluorescence.
Phase contrast microscope
It is extremely valuable for studying living unstained cells. It uses a conventional light microscope fitted with phase contrast objective and phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell which differ only slightly in their refractive indices or thickness.
As light passes through a medium other than vacuum, interaction with this medium causes its phase to change in a way which depends on properties of the medium. These changes in phase carry large amount of valuable information. However, these changes in phase are not easily observed by human eye. Therefore, optical mechanism is employed to translate variation in phase into corresponding change in brightness of structures and hence is detectable by eye.
Illumination produced by tungsten halogen lamp is focused on a specialized condenser annulus positioned below sub stage condenser. Light passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures present in specimen. Undeviated and diffracted light collected by the objective is segregated by phase plate and focused at the intermediate image to form final phase contrast image.
Microscope is the instrument most characteristics of the microbiological laboratory. The magnification it provides enables us to see microorganisms and their structures otherwise invisible to the naked eye. The magnification attainable by microscope ranges from X100 to X400, 000. Several different kinds of microscopy are available many techniques have been developed by which specimens can be prepared for examination. Each type of microscopy and each method of preparing specimen offers advantages for demonstration of specific morphological features.
There are two fundamentally different types of microscope. Light microscope and electron microscope. Light microscope uses a series of glass lenses to focus light in order to form an image. Maximum magnification attained is X1, 500. Electron microscope uses electromagnetic lenses to focus a beam of electrons. Maximum magnification attained is X400, 000.
Both living and dead specimens can be viewed with light microscope and often in real color.
Only dead microorganisms are viewed with electron microscope and never in real color.
Magnification is not the best measure of a microscope. Resolution or resolving power, the ability to distinguish two adjacent points as distinct and separate in a specimen, is much more reliable estimate of a microscope’s utility. Greater magnification without greater resolution i.e., mere increase in size without the ability to distinguish structural details is not beneficial. The largest magnification produced by a microscope may not be the most useful because the image obtained may be unclear or fuzzy. Magnification beyond the resolving power is of no value and is called empty or useless magnification. The resolving power of a microscope is a function of the wavelength of light used and the numerical aperture of the lens system. Total magnification of the system is determined by multiplying the magnifying power of the objective by that of eyepiece.
Resolution of light microscope is 0.5 µm approximately.
Resolution of electron microscope is upto 1 nm
In bright field microscopy, the microscopic field is brightly lighted and microorganisms appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light but staining them with dye greatly increases their light absorbing ability resulting in greater contrast and color differentiation.
All modern light microscopes are made up of more than one glass lens in combination. The major components are condenser lens, the objective lens and the eyepiece lens. Each of these components is in turn made up of combinations of lenses which are necessary to produce magnified images.
There are two basic types of compound light microscope stand- upright and inverted microscope. Upright microscope is for viewing specimens. Condenser lens and light source are below specimen. Stage is movable.
Inverted microscope is for manipulation of specimen directly on stage. E.g,. microinjection of macromolecules into tissue culture cells, invitro fertilization of eggs etc. condenser lens and source of light are above specimen. Objective is movable.
Numerical aperture is a measure of the ability of lens to collect light from the specimen. Lenses with low numerical aperture collect less light than those with higher numerical aperture. Higher numerical aperture objective yields best resolution. The angle theta subtended by optical axis and the outermost rays still covered by objective lens is the measure of aperture of objective. It is half aperture angle.
The magnitude of this angle is expressed as a sine value.NA=n X sin theta
Sin theta=sine value of half aperture angle
N=refractive index of the medium filling the space between front lens and coverslip.
For dry objective n=1=refractive index of air
For oil immersion n=1.56=refractive index of oil immersion which is equal to refractive index of glass.
The wavelength of light used in optical microscope is also limited. The shorter the wavelength of illuminating light, the higher is the resolving power of the microscope. Visible wave length of light rages between 400 nm and 750 nm. By using ultraviolet light as light source, the resolution can be improved.
Greatest resolution using visible light=200 nm
Greatest resolution using ultraviolet light=100 nm
Greatest resolution in light microscopy is obtained using shortest wavelength of visible light and objective with maximum numerical aperture.
Dark field microscopy
It produces image of brightly illuminated objects on a black background. Brilliant illumination of objects is accomplished by equipping light microscope with a special kind of condenser that transports a hollow cone of light from the source of illumination. Most of the light directed through the condenser does not enter the object. Therefore, field is dark. However, some of the light rays will be diffracted (scattered) if transparent medium contains objects such as microbial cells. This diffracted light will enter the objective and reach the eye. Thus objects or microbial cells appear bright. It is used for viewing motility and outlines of objects in liquid media such as living spermatozoa, microorganisms, cells growing in tissue culture.
Spirochaetes Treponema pallidum causative agent of syphilis STD can’t be seen in Gram stained smears, Borrelia, Leptospira, Vibrio.
Fluorescence microscopy
In fluorescence microscopy, ultraviolet light which has very short wavelength and is not visible to the eye, is used to illuminate organisms, cells, particles which have been previously stained with fluorescing dyes called fluorochromes. These dyes are able to transform the invisible short wavelength ultraviolet light into longer wavelength visible light. The fluorescent stained particles appear glowing against a dark background.
Two types of fluorescence microscopy are used in medical laboratory work- transmitted light fluorescence (TLF) and incident light fluorescence (ILF) also called epifluorescence.
Transmitted light fluorescence –Short wavelength light from a fluorescence lamp such as mercury vapor or quartz halogen lamp passes through primary or excitation filter which removes all unwanted color or wavelength of light and passes only those that are required. The transmission of this filter must match the emission peak of the fluorochromes being used. The light is then brought to the specimen by a dark field immersion condenser. The fluorescence which is given off by the specimen passes through a secondary or barrier filter located between the objective and the eye which filters of all the light other than fluorescence wavelength specific to the specimen
Incident light fluorescence
This involves illuminating the specimen fro above (epifluorescence) using a dichroic mirror which reflects selectively the shorter wavelength radiation and transmits the longer wavelength fluorescence i.e., it is transparent to wavelength above given value and opaque to those below.
The light passes through an excitation filter and is directed onto dichroic mirror located above specimen. The mirror reflects short wavelength excitation light on specimen. The visible light from the reflected specimen passes back through the objective to the dichroic mirror which transmits the longer fluorescent wavelength. A barrier filter ensures that only the fluorescence wavelength specific for the specimen reach the eyepiece. No condenser needed for incident light fluorescence.
Phase contrast microscope
It is extremely valuable for studying living unstained cells. It uses a conventional light microscope fitted with phase contrast objective and phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell which differ only slightly in their refractive indices or thickness.
As light passes through a medium other than vacuum, interaction with this medium causes its phase to change in a way which depends on properties of the medium. These changes in phase carry large amount of valuable information. However, these changes in phase are not easily observed by human eye. Therefore, optical mechanism is employed to translate variation in phase into corresponding change in brightness of structures and hence is detectable by eye.
Illumination produced by tungsten halogen lamp is focused on a specialized condenser annulus positioned below sub stage condenser. Light passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures present in specimen. Undeviated and diffracted light collected by the objective is segregated by phase plate and focused at the intermediate image to form final phase contrast image.
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