Practical Physiology N Geetha
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Compound MicroscopeChapter 1

Fine structural organization of tissues can be appreciated only by the use of microscopes. Robert Hook made the first compound microscope in 1695. It has been made perfect by others. Microscopy works on the principles of optics.
Microscopy is of two types: light microscopy and electron microscopy. Light microscopy involves the use of either artificial light or natural sunlight. Electron microscopy involves the use of a beam of electrons, which, by virtue of refraction/diffraction or absorbance through a series of converging/diverging electrostatic plate lenses or magnets, renders the view of the object enlarged.
Light microscopes include simple microscope and compound microscope.
Simple microscope works on the principle of simple biconvex lens. The image formed is virtual and erect. The magnification in simple microscope ranges from 2 to 20 times. They do not give a three-dimensional appreciation of the object. They are used in preliminary screening examination of the object.
Compound microscopes are called so because they are combinations of a double set of lenses referred to as objective lens that lies in proximity to the object and eyepiece lens which lies near the observer's eye. The eyepiece and objective lenses have different magnification. Compound microscopes include monocular compound microscope having single eyepiece, binocular compound microscope that has two eyepieces, phase contrast microscope, polarizing microscope, fluorescence microscope, etc. Monocular compound microscope is discussed in detail.
Parts of the Compound Microscope
Compound microscope consists of:
  1. Mechanical parts
  2. Optical parts
The mechanical parts consist of the stand and the arm. The optical parts consist of eyepiece and the nosepiece that carries the objectives (Fig. 1.1).4
zoom view
Fig. 1.1: Compound microscope
Mechanical Parts
The stand that gives stability to the microscope consists of a heavy foot or base and a C-shaped limb or handle that contain the optical parts. A hinged joint that helps to tilt the microscope to any angle convenient for the observer to view the object connects the limb and the foot. The microscope can be inclined a little so that the person looking into the microscope need not bend. This is useful if prolonged continuous work is to be done as in pathology departments to view histology slides. If fluid preparations are to be examined the stage must be placed horizontal. For example, while doing RBC count, WBC count, etc. if the microscope is kept tilted the fluid will drain away from the counting chamber.
The arm carries the body tube in its upper part and the stage and the sub-stage in its lower part.
  1. Body tube: Body tube is fitted at the upper end of the handle. The optical parts are held in position by the body tube that can be moved up and down by means of coarse and fine adjustment screws one pair on either side. The coarse adjustment screw is identified by the bigger size of the knob while fine adjustment has a smaller knob. When one coarse or fine adjustment screw is turned, the one on the other side also rotates at the same time. Coarse adjustment moves the tube rapidly through a large distance when the adjusting screw is rotated. The fine adjustment works in a similar way but several rotations of the screw head are required to move the tube through a very small distance, i.e. one rotation is equal to 0.01 mm or less. Thus the height of the tube can be adjusted so that the objective lens can be kept at its optimal working distance from the object. (The body of the microscope may be of two types. In one type, the stage is fixed and the body tube can be moved up or down; while in the other type, the body tube is fixed and the stage is moved up and down.)
    5The body tube is 16–17 cm in length and is called the tube length. It is the distance between the upper end of the objective and the upper-end of the eyepiece. The distance between the lower focal point of the objective and the upper focal point of the eyepiece is called the optical tube length, which is about 25 cm. Increase in the tube length increases the magnification but this may result in loss of clarity of the image. It is through the body tube that light passes to the eyepiece.
  2. Stage: Stage is a rigid platform on which the slide containing the specimen to be examined or, the counting chamber is kept. It consists of a fixed stage and a mechanical stage. The fixed stage is a square platform with an aperture in its center to permit light to reach the object. Mechanical stage is a calibrated mechanical frame located on the right edge of the fixed stage. It consists of a slide holder that keeps the slide in position. There are two screws for moving the slide forwards, backwards, and from side to side.
  3. Sub-stage: The sub-stage lies below the stage. It carries the condenser and the iris diaphragm fitted in a short cylinder. Sub-stage can be lowered and raised with the help of a screw.
    The sub-stage condenser consists of two lenses that are corrected for spherical and chromatic aberration and it is used to focus light on the object to be viewed. It can be raised or lowered and thus the focus of light can be changed. It also helps in resolving the image. The full resolving power of the objective is possible only by the use of a proper condenser system. An ideal condenser system is an optically inverted objective. It is constructed in such a way that it gives a solid cone of rays that reaches the object in a perpendicular direction.
    The numerical aperture (NA) of a lens is the ratio of its diameter to its focal length. As the numerical aperture increases, the resolving power of the lens also increases. The condenser has a fixed NA for a particular amount of light passing through the lens. Decreasing the amount of light passing through the lens can decrease the numerical aperture. Therefore the illumination has to be increased as the objectives are changed from low power, to high power, or to oil immersion. Raising or lowering the condenser can vary its numerical aperture. While viewing under a particular objective, the NA of condenser and the objective should be the same.
    All the light passing through the condenser is collected by the objective and helps in maximum clarity of the object viewed. So the position of the condenser must be changed with each objective to get the light focused and to increase the resolving power of the microscope. The proper use of the condenser is necessary for obtaining the maximum resolution of an image.
    The iris diaphragm is seen below the condenser and is attached to it. Function is to control the amount of light reaching the object. It can be opened or closed with the help of a lever on its side. By narrowing the aperture of iris diaphragm, the numerical aperture of the condenser can be decreased.
Illumination System
Below the condenser a double-sided reflecting mirror is attached which is plane on one side and concave on the other side. It can be tilted in all directions. A microscope can function optimally only if proper illumination is there. All the light reaching the eye of the observer should come from the object. In the microscope, proper illumination is provided by the position of the 6condenser, mirror, and the size of the iris diaphragm. The source of illumination can be sunlight that is collected by the mirror, or it can be an inbuilt electric microscope lamp for all powers of the objectives. A frosted tungsten lamp provides uniform white light. If the light source is inbuilt, there is no need of the mirror. Here the intensity of light can be adjusted with a knob provided at the base in front.
Optical Magnifying Parts
Eyepiece is fitted at the top of the body tube. The height of the eyepiece varies—5× is the tallest and 15× is the shortest one. 5× and 10× eyepieces are commonly used (× denotes the magnification, i.e. 5× means the image reaching the eye piece is magnified 5 times). 6×, 8×, and 15× eyepieces are also available. Each eyepiece has two planoconvex lenses, one at the top and the other fitted at the bottom. The lower lens called the field lens collects the divergent rays of the image magnified by the objective and passes them to the eye lens that further magnifies the image. A pointer eyepiece has a small pin mounted in it. It is used to point out a specific object in a field. A demonstration eyepiece has two separate eyepieces mounted on a horizontal barrel. Two people can simultaneously observe the mounted object.
Nosepiece is fitted at the lower-end of the body tube. It has two parts, fixed nosepiece and revolving nosepiece. The revolving nosepiece carries three objectives lenses whose magnification power is 10×, 40×, and 100×, respectively. The desired objective can be rotated into position and the correct position is indicated by a click sound. The aperture of the low power lens is the largest and it has the maximum focal length. The oil immersion lens has only a pinhole aperture. The oil immersion lens has a very short focal length and if the aperture is large, spherical, and chromatic aberration will distort the image.
Image Formation by the Microscope
Image is formed with the help of two convex lenses, the objective lens and the eyepiece lens. The objective lens forms a real inverted and magnified image of the object, which is further magnified by the eyepiece lens. The function of the field lens of the eyepiece is to collect the diverging rays of the primary image formed by the objective lens and pass it to the eye lens of the eyepiece. A virtual magnified image of the object is seen through the eyepiece. The image appears to be at a distance of 25 cm in front of the eyes.
Working Distance
The working distance is the distance between the objective and the slide under study. The distance decreases with increasing magnification. An objective operates at a distance from the object that is roughly equal to its focal length. Focal length is maximal for low power lens and minimum for oil immersion lens. The working distance is 10–13 mm for low power, 1–3 mm for high power, and less than 0.5 mm for oil immersion lens, respectively.
Magnification of the Microscope
Magnification power of the microscope is the degree of image enlargement. The magnification produced by the microscope is obtained by multiplying the objective magnification by the eyepiece 7magnification. For example, if the eyepiece used is 5×, and the objective is 10×, then the total magnification is 5 × 10 = 50. That is, the image of the object is magnified 50 times. The maximum magnification in compound microscope is obtained with an oil immersion objective that gives a total magnification of 1,000 times. The maximum resolution of the magnified image, by the most powerful light microscope, is limited by the wavelength of the light source used. Further magnification, therefore, can be obtained only by reducing the wavelength of the light source.
Resolving Power
Resolving power of the eye indicates the capacity of the eye to differentiate between two points kept close to each other as separate and distinct from each other. The resolving power of unaided human eye is between 0.15 mm and 0.25 mm. The resolving power of lens depends on its NA as well as the wavelength of incidental light (λ). The resolving power of a microscope is expressed in terms of limit of resolution (LR) or the minimum separable distance. If the distance between two points is less than LR, the two points appear as one. The limit of resolution of a standard light microscope is around 200 nm. The electron microscope gives very high magnification and has a resolving power of 0.5 nm.
zoom view
Taking into consideration the resolving power of the eye, only a magnification capable of separating 1/100th of an inch would be useful. Further magnification will cause strain to the eye.
Adjustments of the Microscope
Low Power Adjustments
  • The low power objective in which is labeled 10× is brought in line with the eyepiece lens.
  • The condenser is lowered.
  • Iris diaphragm is half opened so that a narrow rim of light rays at the periphery is cut-off.
  • If mirror is used, concave mirror is turned toward the source of light.
  • Look at the lower end of the objective by bending by the side of the microscope and by coarse adjustment bring down the low power objective near the slide, but take care that the objective does not touch the slide.
  • Then look into the microscope through the eyepiece and slowly raise the objective by using the coarse adjustment screw till the object can be clearly seen.
  • The cells and their components are three-dimensional structures and lie at different levels. So under any magnification, the focus should not be kept fixed but continuously use the fine adjustment screw to see the details of the object. This process of continuously adjusting the fine adjustment screw to bring the various structures into view clearly into and out of focus alternately is called racking the microscope.
High Power Adjustments
  • High power objective, which is marked 40× or 45×, is brought into position and confirmed by the click sound.
  • Condenser is half raised.
  • 8Plane mirror is turned toward the source of light if mirror is present.
  • Iris diaphragm is 3/4th opened.
  • As for low power adjustment, raise the body tube very slowly till the object is clearly seen. While viewing under high power, the working distance is very little. So it is better to do the adjustments using the fine adjustment screw.
  • While looking through the eyepiece, the objective should never be brought down with coarse adjustment screw. This may lead to damage of the costly objective lens.
Adjustments for Oil Immersion
  • Oil immersion objective, which is labeled 100×, is brought in line with the eyepiece lens.
  • Condenser is raised fully.
  • Iris diaphragm is fully opened.
  • Plane mirror is used.
  • After keeping the slide on the stage, put a drop of cedar wood oil on the slide over the area to be observed.
  • Looking through the side of the microscope, bring down slowly the oil immersion objective till it just touches the oil.
  • Look through the eyepiece and by fine adjustment focus the image accurately.
Care of Microscope
  • Always keep the microscope in its case when not in use in order to protect it from moisture and dust.
  • Place it on the table in a stable position. Take care of the eyepiece when it is removed from the tube.
  • Always keep the microscope clean and free from dust.
  • Cedar wood oil should be removed from the oil immersion lens immediately after completing the experiment. Otherwise it may seep into the body of the objective and damage the lens. The oil should be removed first with a dry soft cloth or with tissue lens paper and then with a little of xylol on it. Use of excess of xylol or any other cleansing agent should be avoided. It loosens the cement material in which the objective is fixed.
  • The surface of the objectives and the eyepieces should be cleaned with soft linen or polishing cloth. Always take care not to touch the glass with fingers and not to blow on them to remove dust.
  • While lifting the microscope, one hand should be placed under the base and the other hand should hold the handle.
  • Each microscope should be thoroughly checked by a mechanic at least once a year.
  • Never bring the objective down using the coarse adjustment screw while looking into the microscope. It may hit the slide and cause damage to the lens, which is costly.
Other Microscopes
Phase Contrast Microscope
Phase contrast microscope uses principles of light refraction and it helps to identify structures that cannot be seen with ordinary light microscope. It works on the principle of interference. Unstained wet preparations can be studied. In this microscope, a special plate is inserted into 9the condenser, which can retard the speed of some light waves. Since the different cells have different refractive indices, this microscope uses these differences to produce an image with good contrast of light and shade.
Electron Microscope
Electron microscope is used to study the ultra-structural details of tissues and cells. In an electron microscope the electron beam of much lesser wavelength replaces the light source. Electromagnetic fields are used in place of glass lenses. A series of electromagnets are used for converging or diverging the beam of electrons. This renders the view of the object as enlarged one. The electron microscope gives very high magnification and can separate dots that are about 0.2 nm apart. There are two types of electron microscopy:
  1. Transmission electron microscopy (TEM) gives a two-dimensional image of the object. It uses a beam of electrons instead of light and electromagnetic field instead of glass lens. Magnification is about 100,000 times.
  2. Scanning electron microscopy (SEM) gives a three-dimensional image. The image is produced in a cathode ray oscilloscope and it can be magnified.
Fluorescence Microscope
A fluorescent dye is used to stain tissues or microorganisms like mycobacterium tuberculosis; lipids, and elastic fibers, etc., which are then studied under this microscope. Ultraviolet light is used for illumination.
  1. What are the adjustments of microscope for low power, high power, and oil immersion?
    Refer to page nos. 7 and 8.
  2. Why oil is used while viewing under oil immersion objective?
    Cedar wood oil is used in oil immersion to avoid the thin layer of air between the slide and the objective so that the glass slide and the objective lens become a continuous column. This avoids refraction of light rays coming from the condenser and allows enough light to enter the objective. When light rays pass from a denser medium (glass slide) to a rarer medium (air) the rays get refracted away from the aperture of the objective. The oil immersion lens has only a pinhole aperture. Any factor that reduces the amount of rays falling on the objective impairs the quality of the image and the image will be faint. Oil removes the layer of air and prevents refraction of light.
  3. Why cedar wood oil is used specifically in oil immersion?
    Cedar wood oil is used because it has the same refractive index as that of glass (1.515) and least refraction of light rays occur. Liquid paraffin and glycerin can also be used but cedar wood oil gives best result.
  4. What are the precautions you should take while using the microscope?
    Refer to page no. 8.
  5. Why the oil immersion objective has only a pinhole aperture?
    The focal length of oil immersion lens is very short. So if it has a large aperture, more light will enter through the periphery of the lens leading to spherical and chromatic aberration, which will distort the image.
  6. 10What is the magnification for low power, high power, and oil immersion when a 10× eyepiece is used?
    Magnification power of the microscope is the degree of image enlargement. The magnification produced by the microscope is obtained by multiplying the objective magnification by the eyepiece magnification.
    Low power – 100 times (objective 10×)
    High power – 400 times (objective 40×)
    Oil immersion – 1,000 times (objective 100×)
  7. What does working distance mean? Give its value for different magnifications.
    The distance between the object and the objective lens in the microscope at which the object is sharply focused is called working distance. The objective working distance decreases as the magnification and numerical aperture increase. It is maximal for low power 10× (5–8 mm), less for high power 40× (0.5 mm), and least for oil immersion 100× (0.13 mm).
  8. What are the functions of the condenser and iris diaphragm?
    Condenser condenses the light rays and focuses them on the object. All the light passing through the condenser is collected by the objective and helps in maximum clarity of the object viewed. So the position of the condenser must be changed with each objective to get the light focused and to increase the resolving power of the microscope. If the condenser is raised too high maximum light reaches the objective lens. The aperture of the low power lens is largest and it will allow large amount of light to pass through. So if the condenser is raised to the maximum while viewing under low power the clarity of the image will be lost due to excessive brightness. So the condenser should be lowered while using low power objective. The rays passing through the condenser system when collected by the objective now suffer from minimum diffraction. The oil immersion objective lens has only a pinhole aperture and so for maximum clarity the condenser should be raised. The proper use of the condenser is necessary for obtaining the maximum resolution of an image. Adjusting the iris diaphragm that is placed immediately below the condenser can control light reaching the condenser.