MICROSCOPE AND THEIR TYPES

MICROSCOPE AND THEIR TYPES

MICROSCOPE

A microscope, in simple terms, is an instrument used to view objects that are not visible to the naked eye.

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The microscope is one of the most expensive and delicate instruments used in a clinical laboratory. Because it can be easily misused, it is important for medical laboratory personnel to be conversant with the working principle of the commonly used light microscope, and should know how to use and maintain it correctly.

cylinder known as the Whenalt cap. 

This cap serves to shape the electron beam. Just beyond the Whenalt cap is the anode which has an aperture through which the electron beam passes. A large voltage is applied between the cathode (the tung. sten filament) and the anode, this gives the electons their high velocity. The wavelength of electron is inversely proportional to their velocity, and therefore they have a very short wavelength (0.1-0.5A/ or 0.01-0.05 nm). The electrons pass through the rest of the microscope without any further acceleration.

cylinder known as the Whenalt cap. This cap serves to shape the electron beam. Just beyond the Whenalt cap is the anode which has an aperture through which the electron beam passes. A large voltage is applied between the cathode (the tung. sten filament) and the anode, this gives the electons their high velocity. The wavelength of electron is inversely proportional to their velocity, and therefore they have a very short wavelength (0.1-0.5A/ or 0.01-0.05 nm). The electrons pass through the rest of the microscope without any further acceleration.

The electron beam first passes through the condenser lens. As in the light microscope, this lens serves to focus the beam to the object, and so provides illumination. The lenses in the electron microscope consist of circular electromagnetic fields. 

The magnetic lens of an electron microscope can have different powers (focal lengths and magnification) depending on the amount of current flowing through the electrical coils. In the light microscope, the lens powers are fixed, but the lenses are movable with respect to the object so that the image can be focussed and proper conditions of illumination obtained. In the elctron microscope, all the lenses are rigidly fixed, but their focal points are variable by changing lens currents. Thus, the illumination of the object is achieved by varying the current in the condenser lens.

The imaging system of the electron microscope usually consists of two lenses-the objective and the projector lens. A third, intermediate lens, may be present between the two. 

This gives three stages of magnification and makes it possible to achieve high magnification in a reasonable amount of space. However, the numerical aperture of an electron microscope lens is very small and the best resolution is about 4 A i.e., 0.3 -0.5 nm (about 500 times better than that of light microscope which is 0.2 um).

The objective lens is placed with its focal point close to the object: Intermediate images are formed between each lens system. The projector throws its image on to a fluorescent screen (Fig. 3.28a) which

Working Principle of a Microscope..

The microscope magnifies the image of the object being viewed through it. An ordinary magnifying glass is referred to as a simple microscope, while a laboratory microscope is referred to as a compound microscope, or a light microscope, or more appropriately, a bright field microscope. The magnification of the object is produced by the combined action of two lenses, the objective lens near the object, and the eye piece lens near the viewer's eye.

Essentially, the bright field microscope consists of a light source, a condenser that focuses rays of light on the specimen, a stage on which a specimen is placed, an objective lens that produces a magnified image of the objects in the specimen, and an eyepiece or ocular lens through which further magnified image of the object can be directly viewed. The specimen to be viewed with the light microscope has to be sufficiently thin so that light can pass through it. 

Some light is absorbed while passing through the specimen, and a contrast may be produced due to differences in light absorption by different parts of the specimen. However, the optical system of the bright-light microscope does not reveal much contrast in the unstained preparation. Therefore, the contrast needs to be enhanced with staining. Terms and Principles commonly used in Microscopy

Reflection When a ray of light strikes a surface at an angle and it bounces back at an angle of equal size, it is said to be reflected (Fig. 3.10). Reflection not only occurs when light passes through air and strikes an object, but also when it strikes an interface between air and glass. Stray reflections inside the microscope interfere with the path of light rays and degrade the sharpness of the image.

Refraction is simply the bending of a light ray from the "normal" when it passes into a different optical medium. A "normal" line is the line perpendicular to a flat surface. Refraction is caused by changes in the speed of light while passing from one medium into another of different optical density. 

When light enters a more dense medium, it bends towards the normal line; when entring a less dense field, light bends away from the normal line (Fig. 3.11). Optical media include glass(such as lenses, filters, slides, coverslips), air, immersion oil, mounting medium etc.

Refractive Index It is the measure of refraction and is measured as

Velocity in medium (km/sec) It is proportional to the density of the medium. Refractive index can also be defined as the relationship between the sine of the incident angle (a) to the sine of the refracted angle (b).as shown in Figure 3.11.

Refractive index of air is 1.0, of water is 1.3 and of glass is 1.5.

 Lenses in an optical system, the lens collect light rays from an object and redirects them to form a sharp, magnified image of the object in the image plane. There are two basic types of lenses used in microscopy-converging or positive lenses and diverging or negative lenses. The converging lens is convex and directs light to a point. The diverging lens is concave and it bends light outward. Several combinations of these two basic types are possible. However, double convex lens is the most common type used in microscopy. 

Principle focus and optical center The center of lens surface on either side of a biconvex lens is called a center of curvature. A straight line joining these two centers is the principal axis. A ray of light entering the lens along the principal axis does not refract and travels along the same line. Rays of light entering a converging lens parallel to the principal axis, however, are refracted towards this axis. The point at which they meet is called the principal 

focus (F). A biconvex lens has a principle focus on each side of the lens. A ray of light entering a converging lens at an angle emerges parallel to the entering ray, and will pass through the center of the lens. Another ray entering similarly from the other surface of the lens also passes through the centre. The point at which these two rays cross is called the optical centre (O) of the lens. The distance between the optical centre and the principle focus is the focal length of the lens (Fig. 3.12).

Magnification The magnification produced by a lens is defined as the ratio of the distance between the lens and the image plane (b), and the distance between the lens and the object (a) as shown in Fig. 3.13.

In simple words, magnification is obtained by dividing the size of the image by the size of the object. In case of a convex lens, the magnification is maximum when the object is placed just outside the principal focus of the lens.

 Defects in lens systems While lenses provide the desirable aspect of magnification, they also have limitations caused by behavior of light. In microscopy, these limitations arise from two causes-the shape of the lens, and the presence of different wavelengths in the white light used. 

These limitations give rise to two defects, namely, spherical aberration and chromatic aberration. These two aberrations are corrected by compounding lenses of varying refractive indices and dispersing abilities to one compound lens.

Spherical aberration is the indistinct or fuzzy appearance of images due to nonconference of rays of light to a common focus (Fig 3.14).

The spherical aberration occurs when the edge of a lens gives a slightly higher magnification than its centre. This results in the loss of contrast, resolution, clarity and overall focus. 

Spherical aberration is a property of those lenses that have less than perfect spherical shape and it increases with increase in the thickness of the biconvex lens. It can be corrected by compounding it with a biconcave lens that brings the image to a sharp focus.

Chromatic aberration is defined as the fuzzy appearance of the image due to non-convergence of rays of white light to a common focus. This is the condition in which the image is surrounded by a multi-colored fringe, with the blue light being slightly more magnified than the red. 

It is caused by splitting of white light into its component colours while passing through a biconvex lens, which acts as a prism. When white light passes through a prism, the light of shorter wavelength, like blue (wavelength 350 nm), is refracted more strongly than that of a longer wavelength such as red (wavelength 700 nm). 

Thus, the light of one colour is projected at a greater magnification than another resulting in the appearance of coloured fringes in the image of the object (Fig. 3.15). 


Chromatic aberration in modern microscopes is controlled by proper combination of lenses. Achromatic lenses are corrected for one colour while the apochromatic are for three colours.

COMPONENTS OF A MICROSCOPE

Basically, a microscope is made up of three parts:

1. The stand which is a support for all the other parts

2. The mechanical components

3. The lens systems Figure 3.16 illustrates parts of a binocular microscopes. 

The Microscope Stand The stand is the framework of the microscope. It comprises the following: 

(a) The tube which holds the objectives and the eyepiece. For most laboratory microscopes three or more objectives are needed and these are screwed into the revolving nose piece. The eyepiece can either be monocular or binocular. It is inserted the upper end of the tube.

(b) Tube length of a microscope is the distance between the eyepiece and objective.

(c) The body of the microscope supports the focusing mechanisms.

(d) The arm gives correct frame to the tube and the body. 

(e) The stage is the platform on which the object is placed.

A circular hole in the centre of the stage allows light from below to pass through. A mechanical stage enables the specimen on the stage to be Moved in a controlled way. It holds the slide in place and moves it systematically either across or along the stage, by the rotation of two knobs, one for each direction. The knobs are usually located to one side of the stage. There may be a venire scale attached to the stage for each movement. 

(F) The sub stage is immediately below the stage and holds the condenser with its iris diaphragm and a holder for light filter.

(g) The foot or the base may be in the form of a large block or horse shoe shape. It enables the microscope to sit firmly on the bench.

The Mechanical Components

The mechanical components include: Coarse adjustments The coarse adjustment is controlled by the rack and pinion mechanism manipulated by a pair of large knobs one on each side of the body of the microscope. By rotating these knobs, the tube with its lenses moves up and down fairly rapidly. It is used for rough focussing which is often enough for low power objectives.

Fine adjustments- The high power objectives require fine focussing of the objects which is made possible with the fine adjustment. The fine adjustment is controlled by two smaller knobs, one on each side of the body. It moves the objective up and down slowly. In some models the fine adjustment is incorporated on the same knob as the coarse adjustment. In some microscopes the fine adjustment knob is graduated in microns to indicate the distance moved. 

Condenser adjustments- The condenser has the features for focusing, adjustments of aperture iris diaphragm) and centering. It is usually focused up and down by rotating a knob attached to one side of it. The iris diaphragm which is just below the condenser is used to adjust the condenser aperture. It is made up of a number of leaves which can be opened and closed by moving a small projecting lever (Fig. 3.17).

Centering- Where the condenser is not permanently fixed, it can be centered to bring illuminating beam of light to strike accurately at the object.

The Lense Systems-

The microscope lense system, which is also referred to as optics of the microscope, comprise the objective, the eyepiece, the condenser and the light source. A mirror may be necessary to direct an external light source to the object.

THE OBJECTIVE- The function of the objective is to produce a magnified image of the object. A light microscope has three or four objectives of varying magnifying power which are fitted on the revolving nose piece. Depending on the size of the object and the magnification required, any one of these objectives can be brought into the path of light by revolving the nose piece. Each objective is a complicated combination of lenses.

Magnifying Power of Objectives

1. Low power objective this can be identified by such markings as 10 X, or 16 mm, or 2/3 inch. This objective can also be coded with a yellow ring. The objective is generally used for rapid scanning of the microscopic field.

2. High dry objective this objective is marked 40 X, or 4 mm, or 1/6 inch. The color code is blue ring. This objective gives highest magnification in dry objectives.

3. Oil Immersion objective this is the objective with highest magnification in the ordinary light microscope. It is marked 100 X, or 2 mm, or 1/12 inch. It is denoted by a white ring. This objective is always used in conjunction with immersion oil. 

Oil, such as cedar wood oil, having the same optical density as glass, is placed between the object and the lens to eliminate refraction of light. The oil used for this purpose is called immersion oil and it enables the light to pass in a straight line from glass through the oil and back to the lens glass as though it were passing through the glass all the way( Fig. 3.18).

Resolving Power

The limit of useful magnification is set by the resolving power of the microscope. Resolving power of the microscope objective is its ability to reveal closely adjacent points as separate and distinct. Quantitatively, it is a capacity to distinguish two neighboring points as separate entities. It depends

2. High dry objective This objective is marked 40 X, or 4 mm, or 1/6 inch. The colour code is blue ring. This objective gives highest magnification in dry objectives.

3. Oil Immersion objective This is the objective with highest magnification in the ordinary light microscope. It is marked 100 X, or 2 mm, or 1/12 inch. It is denoted by a white ring. This objective is always used in conjunction with immersion oil. Oil, such as cedar wood oil, having the same optical density as glass, is placed between the object and the lens to eliminate refraction of light. The oil used for this purpose is called immersion oil and it enables the light to pass in a straight line from glass through the oil and back to the lens glass as though it were passing through the glass all the way( Fig. 3.18).

Resolving Power

The limit of useful magnification is set by the resolving power of the microscope. Resolving power of the microscope objective is its ability to reveal closely adjacent points as separate and distinct. Quantitatively, it is a capacity to distinguish two neighbouring points as separate entities.

It depends largely upon the angle of light entering the objective and the refractive index of the medium between the object and the objective. The presence of oil between the object and the objective conserves many of the light rays which, would otherwise, have been lost by defraction.

Resolving Power ( 0.61 x 1

NA where 2 is the wavelength of light used and NA is the numerical aperture. For green light, where lambda is approx. 540 nm, and numerical aperture of the lens used is 1.4, the resolving power is about 200 nm (0.2um).

This means that the minimum distance between the two points, to be seen separate and distinct, should be 200 nm (Fig. 3.19a). If it is less than this, they will appear as a single object (Fig. 3.19b). When further magnification of the two points fails to show them separate and distinct, it is called empty magnification.

Numerical Aperture-The numerical aperture (NA) is defined as the product of the refractive index of the medium outside the lens (n) and the sine of half the angle of the cone of light absorbed by the front lens of the objective (u). This is expressed mathematically as

Figure 3.19 shows NA objective with-out oil The refractive index of air is 1 while that has a higher NA then any objective

Some objectives have the numerical aperture engraved on them. The numerical apertures of the commonly used objectives are shown in Table 3.1 below.

The numerical aperture is in many ways more important than the magnification. This is because an increase in NA results in an increase in resolution.

Focal Length and Working Distance

The focal length of a lens is the distance between the optical centre and the point at which the parallel rays of light passing through it come to a critical focus. Since it is not possible to determine the exact optical centre of the objective, the term equivalent focal length is used. 

The focal length, however, is of less practical value than the working distance which is the distance between the front lens of the objective and the object in focus. The working distance is much less than the focal length and it is of practical importance, especially with high power objectives where the use of a wrong working distance can result in the damage to the front lens of the objective. To reduce such damage to a minimum, spring loaded objectives are in use.

In modern microscopes, objectives are par-focal. This means that when an objective is used to focus on an object, the object will still be in focus with a small touch of the fine adjustment when another objective is swung into position. This prevents the possibility of damaging the object while changing from a low power to a high power objective during focussing. It also prevents the lens of the objective from being scratched.

Double achromatic lens is a composite of two lenses used to correct spherical and chromatic aberrations in the lens. The first lens is a convex lens and is of crown glass, and the other is a concave lens and is made of flint glass.

The quality of an objective depends on how well it has been corrected. In most achromatic objectives, chromatic aberrations are corrected for two wavelengths while the spherical aberration is corrected for one wavelength. Achromatic objectives are most widely used in routine microscopy.

Types of Objective

Apochromatic lens (objective) is a highly corrected objective. These objectives are costly and are used mainly for research work. They are corrected for three spectral colours for chromatic aberrations, and two spectral colours for spherical aberrations. Apochromatic lenses are meant for oil immersion or high powered objectives and are 
used in conjunction with a compensating eyepiece 

Fluorite objectives are also known as semiapochromatic objectives. They are intermediate between achromatic and apochromatic objectives. The function of a fluorite objective is to correct the two types of aberrations for two wavelengths each,thus increasing the value of the numerical aperture for greater resolution.

The objectives are particularly useful in the visual or photographic recording of fine detailed demonstration of the tubercle bacilli following Ziehl-Neelsen staining. A 50X fluorite objective has a wider field than 100X oil immersion objective and a sharper image than a high power dry objective.

 Flatfield (plano) objectives are corrected for field curvature. Additional lenses are built into this objective to flatten the image across the field so that the entire field is simultaneously focussed. Flatfield objectives are mainly used in photo-microscopy.

Spring loaded objectives are found in modern microscopes and are made to protect the specimen and the front lens of the objective from being damaged during focussing. The front mount of the objective pushes in when pressed against the slide.

THE EYEPIECE OR THE OCULAR SYSTEM

The eyepiece lens magnifies the primary image produced by the objective lenses and presents the final image of the object to the eye. The range of eyepiece magnification availble is 5X, 7X, 10X, 15X or 20x. 

The eyepiece with higher power gives greater magnification, but a brighter and sharper image is obtained with a low power eyepiece. Therefore, 10X eyepiece is the most commonly used eyepiece. Some eyepieces have built-in pointers so that specific features within the field of view may be indicated. This is especially useful for teaching purposes.

Types of Eyepieces

Like the objective, the eyepiece is also a complex system of more than one lenses. Depending on the arrangement and type of lenses used, they can be described as:

Huygenian eyepiece The most common type of eyepiece is the Huygenian eyepiece. It is normally used with achromatic objective. It consists of two planoconvex lenses of different sizes both facing the objective. 

1.The lens closer to the eye is called the eye lens and the other, field lens. At the focal plane of the eye lens, and between the two lenses, is the diaphragm (Fig. 3.21).

2. Rhamsden eyepiece This eyepiece also consists of two lenses, but instead of facing the objective as in Huygenian eyepiece,they face each other.

3. Compensating eyepiece This type of eyepiece is intended for use with apochromatic or fluorite objective, but not with achromatic objective.

 
4. Wide-field eyepiece This is used in association with flat field objectives. It is particularly useful when there is a need to scan a wide area of specimen. 

5. Monocular and binocular microscopes A microscope with only one eyepiece is referred to as monocular microscope and one with two eyepieces is called a binocular microscope. In the modern binocular microscope, the rays of light reflected from the object are equally divided between the two eyepieces. This is achieved by the use of prism called the swan cube (Fig. 3.22). 

The binocular microscope has the advantage over the monocular type that the image can be viewed with both eyes and therefore causes less strain to the eyes during long periods of microscopic work.

A trinocular head attachment is an inclined binocular eyepiece with a third protruding tube and sliding prism. This additional tube is used for attaching a camera for photomicrographic work.

6. Measuring eyepiece An eyepiece fitted with a disc which has a scale on it (graticule) is called a measuring or micrometer eyepiece. By adjusting the upper lens, the graticule can be sharply focussed on the object.

The measurement of each division of the graticule for each objective can be calculated by using a stage micrometer which is a measured scale. Details of measurement of the object using calibrated eyepiece scale are given later under micrometry.

CONDENSER AND IRIS DIAPHRAGM

The condenser is a large lens mounted below the stage, with the iris diaphragm below it. It consists of two or three lenses, and allows light to pass through to the objective at a sufficiently wide angle to illuminate the object uniformly at the point of focus. It supplies the object with a cone of light of correct size and character in order to achieve the maximum performance of the system.

Abbe condenser For ordinary transmitted light microscopy, the Abbe condenser is mostly used. The illumination it provides is called Abbe illumination. It is made of two large planoconvex lenses separated by a layer of air and are provided with an adjustible iris diaphragm that is mounted at the back focal plane of the lens. Optically speaking it

is most efficient and it is easy to use since it does not require to be focused or centered. It is employed where high performance is essential. 

Condenser aperture The condenser, like the objective, has numerical aperture (NA). This NA is the angle of the cone of light that passes from it through the object on to the objective. The full NA of the objective can only be achieved if the condenser supplies a solid cone of light of similar aperture. 

The NA of the objective, therfore, should match the NA of the condenser. Using too large a condenser aperture, however, results in the production of a glare (too much light entering through the condenser up to the eye of the observer) which distorts the image of the object.

The iris diaphragm is used to regulate the angle of cone of light entering the condenser.

Condenser with a swing-out upper lens This type of condenser has a top upper lens which can be swung aside when a low power objective is used. It thus makes it possible for light to fill a much larger field of view of low power objective. The substage may be lowered slightly.

Aplanatic condenser When an Abbe condenser is found to be inadequate, the aplanatic condenser is usually the answer. It is well corrected for spherical aberration.

Achromatic condenser This type of condenser is corrected for both chromatic and spherical aberration. It is mainly used for critical work. It is very expensive.

FILTERS

Light filters are required in the microscope for sev. eral reasons, such as:

1. To reduce the brilliance or intensity of light For example, neutral grey filter.

2. To increase contrast and resolution. For example, blue and green filters.

3. To adjust the colour balance of light to give the best visual effect.

4. To provide monochromatic light when required.

5. To absorb heat from the high intensity lamps.

6. To transmit light of a selected wavelength. or example, an exciter filter used in fluorescence microscopy.

7. To protect the eye from injury by ultravioletrays. For example, barrier filter used in fluorescence microscopy.

SOURCES OF ILLUMINATION (LIGHT)

The source of light often depends on whether electric current is available or not, and whether the microscope is being used in day time or after dark. It is important, however, that no matter the source, the light should fill the field of view and fill the whole of the back lens of the objective so that the image can be properly viewed.

Daylight Direct sunlight is not very good for the microscope and the eye. It is best to use reflected day light. This is sufficient for use with monocular microscope and it is too weak for use with oil immersion objective.

Electric light For most routine work, a 60 watt electric bulb placed about 45 cm from the microscope is an adequate source of illumination. Quartz halogen and tungsten lamps are widely used. Many modern microscopes, especially the binocular ones, are provided with a built-in source of illumination. 

Battery lamp In the absence of electric light, a 6 volt microscope lamp can be connected to a 6 volt battery. This needs to be used through a transformer.

KOHLER ILLUMINATION

In the past, microscopists talked about critical illumination which is today referred to as Kohler illumination. Kohler illumination gives the highest possible resolution and useful magnification to any objective. Thus it provides uniform illumination which just fills the field of view of the eyepiece.

Requirements for Kohler Illumination

1. A lamp of high intensity but of small light emitting area mounted on a centring mount or some pre-focus mount to ensure that the centre of the filament is on the optical axis of the lamp.

2. A focussable lamp condenser with a multilens system to correct the basic aberrations.

3. An adjustible iris diphragm.

-To Set up Kohler Illumination

1. Focus on the specimen with the lower power objective.

2. Raise the condenser to its highest position.Close the iris diaphragm. Move the condenser down until an image of the iris diaphragm appears.

3. Bring the centre of the circle of light to the centre, using the condenser centring screws.

4. Open the iris diphragm slowly till the circle of light fills the entire field.

5. Remove an eyepiece, and adjust the iris

diaphragm to cover 75 per cent of the illuminated circle. This represents the objective lens aperture. Replace the eyepiece and continue microscopy.

MIRROR

In microscopes without a built-in source of illumination, a mirror is placed below the condenser and iris diaphragm. It is circular and will stay in place when turned in any direction. It reflects the beam of light from the source through the iris into the condenser. It usually comprises two mirrors mounted back to back, with one being flat and the other concave. The concave side is not to be used with the condenser; it is only the flat side that is used in conjunction with the condenser.

TOTAL MAGNIFICATION OF A MICROSCOPE

In the compound microscope, the magnified image is produced by two sets of lenses-the objective and the eyepiece. The objective produces a real, magnified, inverted image of the object which is brought into focus by the field lens of the eyepiece. This primary image when viewed by the eye lens is within the focal length of the lens and a magnified virtual image is produced (Fig. 3.23).

The total magnification of the microscope is thus the magnification of the objective multiplied by that of the eyepiece. For example, using a 40X objective and a 10X eyepiece, the object will be magnified 400 times. If a 100X objective is used with a 10X eyepiece, it will give an increased magnification of 1.000X. 

When this increased magnification results in bringing further details of the object into view, it is called useful magnification. If the enlargement of the object fails to bring into view any more detail, and looses the sharpness of the image, it is called empty magnification (Fig. 3.19b).

Resolving power of the microscope plays a major role in increasing the useful magnification of the microscope. It should be noted that because of the combined action of both the lens systems of the compound microscope, the objective and the ocular, the final image seen is upside down and reversed. The left side appears as the right, and the top as the bottom, and vice versa. This should be borne in mind while moving the object under observation.

USE AND CARE OF A BRIGHT-FIELD MICROSCOPE

A microscope is a very delicate and expensive instrument. It must, therefore, be handled with care. The following points are to be borne in mind while using the microscope.

1. Always lift and carry the microscope wellsupported with hands.

2. Protect the microscope from dust, moisture and direct sunlight.

3. Place the microscope on a firm surface so that it does not vibrate.

4. The user should be seated at the correct height for the convenient use of the microscope.

5. The user must be seated away from direct sunlight.

6. Use the flat side of the mirror if the microscope has no built-in source of illumination.

7. The underside of a glass slide should be completely dry before it is placed onto the stage.

8. The specimen should be viewed with or without oil depending on the type of objective employed.

9. It is always advisable to start viewing with the low power objective (10X). 10. Look at the objective from the side when

lowering it on the object. Then look into the eyepiece and raise slowly to focus.

Note

(a) The working distance for the low power objective is 16 mm, for the dry high power objective is about 4 mm and for the oil immersion objective it is about 2 mm.

(b) When using the low power objective, the amount of light entering the lens should be reduced by lowering the condenser and partially closing the iris diaphragm.

(c) For the high power objectives, the condenser should be raised and the iris diaphragm opened to allow in the required amount of light.

11. If the objective lens is smeared or dirty, it should be cleaned with a clean piece of soft linen or lens tissue. Little xylene may be used if the dirt is difficult to remove. Never use alcohol because it can dissolve the cement used to keep the lenses together.

12. While simple cleaning of the microscope

can be undertaken by an experienced user, major cleaning or maintenance should be done by trained personnel.

13. At the end of the working day, all the objective lenses must be wiped clean and the microscope covered with its protective cover.

MICROSCOPY

Microscopy is the use of microscopes in all their various forms. Although the bright-field microscopy is most commonly used, the basic principle of light microscopy can be modified to perform different functions, for example, dark-field, fluorescence, phase-contrast or electron microscopy. Each of these techniques is capable of performing the basic function of the microscope, that is, magnification. In addition, they have specialised functions such as observing biochemical processes as they occur in a living cell, or a several hundredfold increase in resolution by the use of electrons instead of light waves. Table 3.2 shows the features of different types of microscopy.

Bright-Field Microscopy

• Bright fieldmicroscopy,
• Dark-Field Microscopy
• fluorescence microscopy
• Phase Contrast Microscopy
• Interference Microscopy
• Polarizing Microscope
• Electron Microscopy
• MICROMETRY

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