The following points highlight the thirteen main types of microscopes. Some of the types are: Simple Microscope 2. Compound Microscope 3. Research Microscope 4. Binocular Dissection Microscope 5. Phase Contrast Microscope 6. Interference Microscope 7. Differential Interference Contrast Microscope 8. Fluorescent Microscope 9. Electron Microscope 10. Transmission Electron Microscope and Others.
Type # 1. Simple Microscope:
1. A simple microscope is a convex lens of short focal length.
2. It is used to form a virtual image of an object placed just inside its principal focus.
3. It is made up of a single convex lens or a combination of lenses which functions as a convex lens.
4. The convex lens magnifies the object and also helps to produce a magnified image of a near object which appears to be at the distance of distinct vision.
5. Under simple microscope the enlarged image (Fig. 143) of the object is formed on the retina of the eye of the viewer.
6. A simple lens can magnify an object only three times OX). For getting a magnification of more than 3X, a combination of several lenses is used. Such a combination of several lenses (called elements) functions as a single convex lens, and a magnification of about 20X can be obtained.
7. Improved simple microscopes, used by the biologists during field work, may magnify an object even up to 100 times. In such microscopes, multielement lenses are used. It consists of a combination of a double concave lens of crown glass fitted between two double convex lenses of flint glass. This is called an aplanarlens or achromatic triplet. Three of such aplanar lenses are cemented together and function as one lens.
A dissecting microscope (Fig.144) is an example of a simple microscope used either for dissecting the material or for viewing the magnified image of the material. It consists of only one lens unit. This lens unit may even be an ordinary magnifying glass.
It is used either for dissecting the material or for less magnifications, i.e., only 6X, 12X or rarely 20X. It is mainly used for embryo separation, taxonomic studies, etc. It has a basal foot and a limb. The ‘stage’, made up of a simple glass plate, is attached to the limb.
For the light adjustment purposes, a mirror is attached to the limb under the stage. Mirror can be moved vertically with the help of an adjustment screw. At the tip of the limb is present a folded arm, on which a lens of definite magnification (6X, 12X, etc.) is fitted. Folded arm is moved to keep the lens in the desired position on the stage.
The material to be viewed or dissected under a dissecting microscope is placed on the stage. The eye is placed close to the lens. Folded arm is tilted to bring the lens over the material. Light is adjusted by the movement of the mirror. Focusing is done with the help of the adjustment screw of this microscope.
Type # 2. Compound Microscope:
A compound microscope (Fig.145) comprises either two (objectives and eyepiece) or three (condenser, objectives and eyepiece or ocular) kinds of lenses. The condenser, located above the mirror and below the stage, collects and focuses the light rays into the plane of the object.
The objectives (10X, 40X, 100X) are mounted on a revolving nosepiece. Each objective consists of a set of elements fused together to work as a single lens. The objectives collect light rays from the object and form a magnified real image at some distance above them (Fig. 146).
The eyepieces or oculars are located at the top of the body tube. The eyepiece usually have 5X, 10X or 15X lenses. The eyepiece magnifies the image formed by the objective (Fig. 146). A microscope with one ocular or eyepiece is called monocular microscope while the one with two oculars is called binocular microscope.
Parts:
1. The instrument is so named because it consists of two or more lens systems (Fig. 145).
2. At the top is present the ocular lens. It can be turned around or may be removed. At the top of ocular lens is written 5X or 10X signifying the 5 times or 10 times magnification, respectively.
3. Just below the ocular is a body tube, the bottom end of which contains a circular piece called nose piece. It contains three lenses called objective lenses. Nose piece can be rotated to change the position of objectives.
4. The flat platform present below the objectives is called stage.
5. On the arm of the microscope are present two knobs named coarse adjustment knob and fine adjustment knob.
6. Out of the three objectives, the shortest is low power objective. It has the largest lens but its magnifying power is least of the objective lenses. On the objective may also be written 10X as on ocular lens. It means if a 10X ocular lens is used the magnification is 10 X 10 – 100 times.
7. The other objective is high power objective. Its magnification is equal to the number written on it multiplied by the power of ocular i.e. 5X or 10X (objective x ocular).
8. The third objective is oil immersion. Generally, it contains a black band around the lower end. A drop of oil is used on the slide at the time of studying with the oil immersion objective. Its magnification can be estimated as ocular x objective.
The use of oil is essential in order to keep the light rays properly aligned with the small objective.
9. Just below the stage is the condenser. Its function is to gather light from the mirror and direct it to the objective lens. Condenser may be lowered or raised by a knob present on one side of the microscope beneath the stage.
10. Condenser contains a shutter called iris diaphragm.
11. Just below the condenser is present a mirror having its one surface flat and other concave? Use the concave surface in the day light. Flat surface of the mirror is used when electric lamp is used.
Precautions:
1. Clean the ocular and objective lenses with lens paper, and do not remove them.
2. While studying an object, learn to keep one hand on the fine focus knob and focus continually up and down.
3. While studying any kind of preparation, do not tilt the microscope.
4. Leave the low power objective in place after finishing all the observations.
5. To examine an object, always use the low power first and then the other objectives.
6. Never allow an objective lens to strike either the stage or a slide while focusing.
7. Use always the fine adjustment with high power objective.
8. All wet-mount preparations should be pre-covered by a cover slip.
9. Avoid the habit to remove the parts of the microscope.
10. Do not use oil immersion objective without oil.
11. Diaphragm should be wide open while using oil immersion objective.
Type # 3. Research Microscope:
1. It is a compound microscope having very fine quality lenses and some additional facilities such as oil immersion lens, built-in illumination, binocular viewing and attachment for photographic camera.
2. The 100X objective is called an oil immersion lens. It can be used only by introducing a drop of an immersion oil between the objective and the cover-glass. If the oil is not used, the Image of the object will appear blurred. The specific gravity of immersion oil is equivalent to glass.
3. In the built-in illumination facility, the mirror of the microscope is replaced by an electric bulb housed in a chamber fitted with a diaphragm.
4. Binocular viewing means instead of one eye the researcher can use both the eyes together for viewing. For this facility, this microscope is fitted with two eyepieces instead of one.
5. Microscopes with attachment for photographic camera are provided with trinocular tubes. Of these three tubes, two are provided with two oculars for binocular viewing while the third tube accommodates a camera for taking photographs.
6. By using 15X ocular and 100X objective, a maximum magnification of 1500 times may be obtained under a research microscope.
Type # 4. Binocular Dissection Microscope:
1. In this microscope two microscopic systems are mounted alongside (Fig. 147).
2. The two sets of eyepieces and objectives of this microscope produce two images which are erected by two prisms present inside the body.
3. Sufficient space is present between the objective and the stage. This helps in the easy dissection and proper study of the material.
4. This microscope is used for doing fine dissections of the specimens and to study their gross mounts because a three-dimensional picture is available due to binocular viewing facility.
5. The maximum magnification obtained by this microscope is 150X.
Type # 5. Phase Contrast Microscope:
1. It was discovered by Professor Frits Zernike of Netherlands. Its commercial production was first started in Germany in 1942. For the recognition of his discovery of phase contrast, Zernike was awarded Noble Prize for Physics in 1953.
2. Only living and unstained cells are studied under phase contrast microscope. It is so because the living cells are not usually coloured (i.e. they are pure phase objects), but the light transmitted by their different structures will have phase differences caused both by variations in refractive index arising from changes in protoplasmic concentration and by differences in thickness.
3. This is a specially designed light microscope in which annular phase plate and annular diaphragm (Fig. 148) are fitted.
4. The annular diaphragm is situated below the condenser. It is made up of a circular disc having a circular annular groove. The light rays are allowed to pass through the annular groove. Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied.
At the back focal plane of the objective develops an image. The annular phase plate is placed at this back focal plane. This phase plate is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular groove. This thick or thin area in the phase plate is called the conjugate area. The phase plate is a transparent disc.
5. With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this microscope. This is obtained by separating the direct rays from the diffracted rays. The direct light rays pass through the annular groove whereas the diffracted light rays pass through the region outside the groove.
6. Depending upon the different refractive Indices of the different cell components, the object to be studied shows different degree of contrast in this microscope.
7. No special preparation of fixation or staining, etc. is needed for study an object under phase contrast microscope. This saves a lot of time of the researcher because a clear picture of unstained or living cells is easily seen under this microscope.
8. Applications of phase contrast microscopy in biological research are numerous.
By this, one can study:
(i) The actual processes of mitosis and meiosis in living cells;
(ii) The actual effects of several chemicals on the living cells;
(iii) The behaviour of several microscopic organisms (e.g. Protozoans) towards various chemical and physical factors and;
(iv) Processes related to permeability of plasma membrane, etc.
Type # 6. Interference Microscope:
1. This is an improved type of phase contrast microscope which displays the interference between light transmitted and that not transmitted through a specimen.
2. The interference colours produced in this microscope indicate differences in refractive index.
3. The light in this microscope is split into two beams, of which one passes through the specimen and the other through a reference beam. The two beams are then made to interfere at the image plane. Areas of the specimen having similar optical paths appear in the image similarly coloured or similarly bright.
4. Differing from the phase contrast microscope, in interference microscope the central wave and the diffracted wave are completely separated while passing through the phase plate.
5. In this microscope, the annular phase plate is fixed below the sub-stage condenser while the phase plate is fixed above the objective lens in the objective. The ocular lens is used for observing the image.
6. This microscope can be used to measure the thickness of the cell and its components. It is also used to determine the dry weight of microscopic objects such as proteins, nucleic acid, etc.
Type # 7. Differential Interference Contrast Microscope:
1. Also called DIC microscope or Nomarski microscope, this is an improved version of interference microscope.
2. In the optical system of this microscope only a single light beam is used.
3. Passing through the object, objective and a special birefringent prism, the single light beam gets divided into interfering beams.
4. A polarizer, analyzer filters and a compensating prism are also present in this microscope.
5. Excellent contrast of edges of cells and their organelles are seen under this microscope, and their visual effect is three-dimensional.
6. Similar to phase contrast microscope, only unstained cells or materials are studied by this microscope.
Type # 8. Fluorescent Microscope:
1. This is an advance type of light microscope in which the specimen is irradiated at wavelengths which will excite the natural or artificially introduced fluorochromes. An optical filter of this microscope absorbs the exciting wavelengths but transmits the fluorescent image which can be studied normally.
2. It is a microscope used for observing fluorescence in cells and tissues. (Fluorescence is a phenomenon in which the wavelength of invisible ultraviolet light is converted into a wavelength of light in the visible range).
3. A fluorescent microscope consists of (i) a lamp, (ii) the optical system, and (iii) a system of observation.
4. Ultraviolet (UV) light is used as a source of illumination in this microscope. For getting UV rays, high pressure mercury arcs lamps, xenon arcs lamps or tungsten halogen lamps are used. A pair of adaptors (complementary filters) are also fitted in it. Since UV rays are harmful to human eye, the ocular lens used in this microscope is made of ordinary glass. This helps in preventing UV rays reaching the eye, but the fluorescence can be observed easily.
5. This microscope can detect fluorescent materials (e.g. dyes) easily in the cells, even if they are present in traces.
6. It is also used in the study of DNA, RNA, proteins and chromosome banding, localization of Y-chromosome in man, detection of heterochromatin and in several studies of immunology.
Type # 9. Electron Microscope:
1. The electron microscope is an instrument which utilizes the short wavelength of an electron beam, rather than light waves, to obtain very high magnification and resolution of minute structures for which a light microscope is inadequate. It contains an electric gun whose beam is refracted and focused onto a specimen by an electron lens system. The image of the specimen is magnified and projected onto a stage or fluorescent screen.
2. On the basis of several earlier researches in Physics, Knoll and Ruska (1931) of Berlin were the first to develop an electron microscope. With the help of their electron microscope, they could magnify objects up to 12,000 times.
By an Improved type of electron microscope developed by Borries and Ruska (1938), they could obtain pictures of 20,000 times magnification. In this die magnification of objective coil was 100 and the projector coil was 200. The resolution of this microscope was 100Å.
The electron microscopes with 4-1 OÅ or even better resolution are now available.
3. A magnification as high as 1, 60,000 times can be achieved by vising intermediate coils between the objective and projector coils of the electron microscope.
4. Some of the basic differences (Fig. 149) between a light microscope and an electron microscope are mentioned in Table 13.1.
Some differences between light microscope and electron microscope:
Light Microscope:
1. Visible light is used in this microscope.
2. Source of illumination is situated at the bottom.
3- For magnification in this microscope the lens system consists of glass lenses,
4. The lenses arc ocular, objective and condenser.
5. The image is either seen with the eye or recorded on a photographic film with a camera in this microscope.
Electron Microscope:
1. Electrons are used in electron microscope.
2. Source of illumination is situated at the top in this microscope.
3. The lens system consists of electromagnetic coils in this microscope.
4. This microscope has projector coils, an objective and a condenser.
5. The image in an electron microscope is either recorded on a fluorescent screen or recorded on a photographic film.
5. Construction:
An electron microscope consists of an electric gun, microscope column, electromagnetic coils, a fluorescent screen and some other accessories described below:
(a) The electron gun is located at the top of the body of microscope. It is the source of electrons. It is made up of a tungsten filament surrounded by a negatively biased shield with an aperture. The electron beam is drawn off through this aperture.
(b) The microscope column or central column is made up of an evacuated metal tube. It protects the person operating the microscope from X-rays that are generated when the electrons strike the surface of the metal tube.
(c) The electromagnetic coils or lenses include projector coils, objective and condenser. In each coil, the coils of electric wire are wound on a hollow metallic cylinder. The magnetic field, produced by passing the electric current through the magnetic coil, functions as a magnifying lens.
(d) The fluorescent screen is used for observing the magnified image of the object. It remains coated with a chemical which, on being excited, forms the image as on the screen of television.
(e) Some other essential accessories of the electron microscope Include high voltage transformers (for developing high voltage current for the electron gun and electromagnetic coils), vacuum pumps (for maintaining high vacuum inside the microscope column), a water cooling system (for prevention from overheating of various parts), a circulating pump, a refrigeration plant and also’ a filter system.
All these parts require elaborate arrangements and contribute to the massive size of the electron microscope.
6. The image formation in this microscope occurs by the scattering of electrons. The electrons strike the atomic nuclei and get dispersed. These dispersed electrons form the electron image. By projecting on a fluorescent screen or photographic film, this electron image is converted into a visible image of the object.
7. The electron beam in this microscope is made by accelerating electrons through a potential difference of from 1-1500kV in an electron gun.
8. Only dried specimens are studied by electron microscope. Living cells cannot be studied with this microscope because they possess water which causes large scale scattering of electrons.
9. Ultra thin sections (10-50 nm thickness), which are more than 200 times thinner than those routinely used for light microscopy, are cut for electron microscopy. These are cut with the help of diamond or glass knives of an ultra-microtome.
Type # 10. Transmission Electron Microscope or TEM:
1. Transmission electron microscope or TEM (Fig. 8) is a form of electron microscope in which the electrons are allowed to be transmitted through the objects.
2. The specimen to be studied is evenly illuminated by a broad beam of electrons at 40-100kV, and the image is formed directly by focusing those electrons which pass more or less un-scattered through the specimen either on a fluorescent screen or on a photographic film.
3. This microscope has a fine resolution of 0.3 nm or less but is usually not suitable for living specimens.
Type # 11. Scanning Electron Microscope or SEM:
1. This is also a form of electron microscope in which an extremely fine beam of electrons is made at 3-30kV for scanning a selected or specific area of specimen. This is actually a combination of the technology of electron microscopy and television electronics.
2. The specimen to be examined is usually a solid object. The secondary electrons, which are emitted from or near the surface, are collected and analyzed. After analysis, they form a signal which modulates the beam of a cathode ray tube (Fig. 151) scanned in synchrony with a secondary beam.
3. The images, formed in this microscope, resemble those seen in a hand lens. They can be magnified about 100,000 times.
4. This microscope is extremely useful for studying surface structures of thick specimens, cells, tissues and membranes.
5. Because the electrons in this microscope form the image by getting emitted from the surface of the specimen, the image provides a three- dimensional appearance. This three-dimensional image is formed by secondary electrons which are first collected, amplified and then used for the image formation on the phosphor screen of a cathode ray tube.
6. At least two cathode ray tubes are present in this microscope, of which one is used for visual observation of the specimen and the other is used for photography.
Type # 12. Scanning Transmission Electron Microscope or STEM:
1. In scanning transmission electron microscope or STEM, the specimen is scanned in the same way as in scanning electron microscope or SEM but transmitted electrons are collected and utilized for the formation of picture on the screen.
2. The main advantage of this electron microscope is that it is fitted with detector systems. Due to this facility, it can separately detect the scattered electrons, un-scattered electrons, and also those electrons which are there as a result of the combination of scattered and un-scattered electrons.
Type # 13. Polarization Microscope:
1. This is a special kind of light microscope fitted with a polarizer and an analyzer.
2. Plane-polarized light is utilized in this microscope for studying the presence of preferentially-oriented constituents, their shape, the direction of their orientation and their refractive index in the cells and tissues.
3. The principle of this microscope is based on the behaviour of some components of cell when observed under polarized light.
4. The polarizer and analyzer, the two main components of this microscope, are either made of nicol prism or a polaroid disc. Above the polarizer is fitted the condenser. The analyzer is located above the objective. A compensator or gypsum plate is also inserted in between the polarizer and analyzer.
5. This microscope is also used for both quantitative as well as qualitative studies in several biological disciplines including cytology, anatomy, histology and pathology.