The following points highlight the top eight types of microscopy. The types are: 1. Compound Microscope 2. Bright Field Microscopy 3. Dark Field Microscopy 4. Phase Contrast Microscopy 5. Fluorescent Microscopy 6. Electron Microscopy 7. Transmission Electron Microscopy 8. Scanning Electron Microscopy.
Type # 1. Compound Microscope:
A microscope is an instrument which makes enlarged image of minute objects near the objective lens. The image is formed as shown in the Fig. 35.1.
The compound microscope has two set lenses. One is known as objective and the other eye piece. These are mounted in a holder commonly known as body tube. The lens system nearest to the specimen is called objective which magnifies the specimen to a definite number of times.
The second lens system is called eye piece which is the nearest to eye. It further magnifies the image formed by the objective. Accurate focusing is attained by a special screw appliance called as fine adjustment.
Type # 2. Bright Field Microscopy:
In bright-field microscopy, the microscope field (the area observed) is bright and the microorganisms appear dark because they absorb some of the light. Normally, microorganisms do not absorb much light, but staining them with dye greatly increases their absorbing ability.
Generally microscope of this type produces useful magnification of about X1000 to X2000. At magnification greater than X2000, the image appears fuzzy. It is also called microscopy by transmitted light (Fig. 35.3).
Type # 3. Dark Field Microscopy:
In this type of microscopy, a dark back ground is produced against which objects are brilliantly illuminated. For this purpose the light microscope is equipped with a special kind of condenser that transmits a hollow core of light from the source of illumination. Thus, if the aperture of condenser is allowed to open completely, and a dark field stop inserted below the condenser, the light rays reach the objects form a hollow core.
Any object within this beam of light will reflect some light into the objective and will be visible. This method of illuminating an object where the object appears self-illuminous against a dark field, called dark-field illustration.
The condensers used are Abbe condenser, paraboloid condenser and cardoid condenser. Dark field microscopy is particularly valuable for the examination of unstained microorganisms suspended in fluid wet mount and hanging drop operations.
Type # 4. Phase Contrast Microscopy:
The phase contrast principle was discovered by Fritz Zernike who was awarded Nobel prize in physics in 1953. According to this principle, light waves have variable character for frequency and amplitude. Human eyes cannot perceive a phenomenon when two light rays have similar amplitude and frequency but different phases (Fig. 35.4).
The phase contrast microscope is an ordinary bright field microscope with two additional plates, namely annular diaphragm and phase shifting plate, which enables the usage forming rings to be phase shifted with respect to others. Annular diaphragm allows only a ray of light to pass through the condenser and then to object.
The phase shifting plate is placed at the rear focal plane of objective lens. This disc has a ring of optical dielectric material on which the ring of light from annular diaphragm is focused. The ring has the property of retarding or advancing the phase of light of a quarter of a wavelength traversing it.
From each translucent or transparent particle in the object, consider a single ray of incident light. From this two rays result, one, the direct (undiffracted) ray comes through the annular diaphragm passes through the objective and focused on phase- shifting ring which either retards or advances the ray one quarter wavelength with respect to second ray.
The second ray is also derived from the incident ray, but modified by being scattered or diffracted in passing around margin of the object.
This ray does not pass through the phase-shifting ring but traverse the other area of transparent disc. Its wavelength is neither retarded nor advanced. Thus, there is one quarter wavelength out of phase. The contrast between the two is called phase-contrast. It is valuable device in wet mounts and hanging drops. It increases visualization of cellular structure and traverse the other area of transparent disc.
Type # 5. Fluorescent Microscopy:
Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances emit light of larger wavelength and lesser energy content.
Such substances are called fluorescent and the phenomenon is termed as flourescence. This is the phenomenon which is applied in fluorescent microscopy. In practice, microorganisms are stained with a fluorescent dye and then illuminated with blue light. The blue light is absorbed and green is emitted.
Blue and green fluorscence can be excited by ultraviolet radiation as well. A high intensity mercury lamp is used as light source which emits white light. Two filters are used, one is the exiter filter which transmits only blue light to pass to specimen and blocks out all other colours; the second is barrier filter which blocks out blue light (excitation radiation) and allows green light (or other light emitted by fluorescing specimen) to pass through and reach the eye (Fig. 35.5).
As shown in Fig. 35.5 a high intensity mercury lamp is used as the light source and it emits white light. The excited filter transmits only blue light to the specimen and blocks out all other colours.
The blue light is reflected downward to the specimen by a dichromatic mirror (which reflects light of certain colours but transmits light of other colours). The specimen is stained with a fluorescent dye: certain portions of the specimen retain the dye, others do not.
The stained portion absorbs blue light and emit green light which pass upward, penetrate the dichromatic mirror and reaches the barrier. This filter allows the green light to pass to the eye and blocks out the resident blue light from the specimen which may not have completely deflected by the dichromatic mirror. Thus, the eye perceives the stained portions of the specimen of glowing green against a black background.
Some suitable fluorescence are: acridine orange, acridine yellow; acriflavine auramine 0; fluorescence titan yellow G; rhencine A, etc.
Type # 6. Electron Microscopy:
In 1931 Knoll and Ruska, German scientists discovered electron microscopy. Von Borries and Ruska (1938) in Berlin constructed first practical electron microscope. The commercial instrument first came in around 1940.
In electron microscope the source of illumination is electron beam. The construction and principle of electron microscope are easily related to those of light microscope. The range of wave length of visible light used in light microscope is 4000 Å – 7800 Å, while with an electron microscope employing 60-80 KV electron, the wave length is only 0.05 Å.
In the instrument as shown in the figure, the electron gun generates electron beam. These electrons are concentrated by other components of electron gun producing a fast moving narrow beam of electron.
Electrons are focused by electromagnetic lenses. Electromagnetic lens consists of wire encased in soft iron casing. When electric current is passed through the coil, it generates an electromagnetic field through which electrons are focused.
There are three general types of electromagnetic lenses. The one is placed between the source of illumination and the specimen. This focuses the beam of electron on specimen functions in a similar manner as that of light microscope. The other two lenses are on the opposite side of specimen which magnify the image in similar fashion as objective and ocular in light microscope.
Type # 7. Transmission Electron Microscopy:
The electron source is commonly a tungsten filament of 30-150 KV potential. The electron beam passes through the centre of ring like magnetic condenser and becomes converged on the specimen.
After being transmitted through the specimen (hence transmission electron microscope (TEM), the magnetic objective focuses the electron into a first (real) image of the object which is enlarged (2000 times). The magnetic projector lens then magnifies a portion of the first image producing magnification upto 240,000 or more.
The final enlarged image can be reviewed by striking a fluorescent screen which makes it visible. The image can also be thrown upon a photographic plate for permanent record. Portions of the photographs may be enlarged four to six times giving the picture in the range of two million times as large as the object.
0 = Magnifies 2 million times → 2 mm diameter
4 million max or 2.5 miles in diameter.
Molecules in the microscope interferes with the movement of electrons. To prevent this, the interior of the microscope is kept in the state of high vacuum, around 10-4-10-6 mm Hg. It is also necessary to have specimen ultra thin. The electron beams have a very poor penetrating power, therefore, only small objects or very thin sections of the specimen can be examined.
Type # 8. Scanning Electron Microscopy (SEM):
Scanning electron microscopes combine the mechanism of electron microscopy and television. SEM became commercially available in early 1960’s and the researchers were Knoll, Von Ardenne, Zworytein etc.
In SEM, electrons are not transmitted through the very thin specimen from below but impinge on its surface from above. The specimen may be opaque and of any manageable thickness and size.
If the specimen is an electron conductor, it needs only to be held on an appropriate support. If it is non-conductor, it is allowed to dry but if moist, freeze dried in liquid nitrogen is necessary. The specimen is then coated with metal vapour (gold) in vacuum.
The electrons originate at high energy (20,000 V) from a hot tungsten or lanthanum hexoboride cathode “gun”. These electrons are sharply focused, adjusted and narrowed by an arrangement of magnetic fields.
Instead of forming a broad inverted cone of rays, in SEM a needle sharp probe (about 5 – 10 mm in diameter) is made. This primary beam (probe) acts only as an exciter of image forming secondary electrons emerging from the surface of the specimen.
The probe scans the specimen like that on a blank TV screen. The probe can impinge on depth and heights with equal speed and accuracy giving great depth of field and producing images with three dimensions. Images are elicited from wherever the probe strikes the metal coated areas of the specimen. Magnification is the ratio of final image to the diameter of area scanned.
Any of the secondary electrons with sufficient energy can emerge from the surface. Those that emerge not too far from the point of impact of the probe can be used to form an image. The useful secondary electrons are magnetically deflected to a collector or detector. Here, they produce a signal that represents at any single moment, only 5-10 mm area or spot of impingement of the probe on the specimen.
The successive signals from the collector are amplified and transmitted to a cathode ray (TV) tube. The scanning beam and TV tube beam are synchronized.
The image scan by the eye on TV screen is thus the sequence of signals representing in araster pattern, the successive areas traversed by the primary probe beam. Exposure may range from a few second to one-half hour or more. The TV image may be photographed, video taped or processed in motion on a computer.