USE AND CARE OF Laboratory Microscope PDF

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USE AND CARE OF Laboratory Microscope...

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LABORATORY INSTRUMENTATION

LABORATORY MICROSCOPE

USE & CARE OF LABORATORY MICROSCOPE

A microscope is the most expensive and important piece of equipment used in laboratories. Microscopy forms 70–90% of the work. WORKING PRINCIPLE OF A MICROSCOPE A microscope is a magnifying instrument. The magnified image of the object (specimen) is first produced by a lens close to the object called the objective. This collects light from the specimen and forms the primary image. A second lens near the eye called the eyepiece enlarges the primary image, converting it into one that can enter the pupil of the eye. Useful magnification The objective provides all the detail available in the image. The eyepiece makes the detail large enough to be seen but provides no information not already present in the primary image formed by the objective. The magnification of eyepiece used should therefore be adequate to enable the relevant detail in the primary image to be seen clearly. Increasing further the magnification will reveal no more detail but only an image that is more highly magnified and increasingly blurred. Note: The range of total magnifications within which details in the object are seen clearly in the image (useful magnification) is usually taken as between 500 and 1 000 times the numerical aperture of the objective.

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LABORATORY MICROSCOPE

The most commonly used microscope for general purposes is the standard compound microscope. It magnifies the size of the object by a complex system of lens arrangement. It has a series of two lenses; (i) the objective lens close to the object to be observed and (ii) the ocular lens or eyepiece, through which the image is viewed by eye. Light from a light source (mirror or electric lamp) passes through a thin transparent object. The objective lens produces a magnified ‘real image’ first image) of the object. This image is again magnified by the ocular lens (eyepiece) to obtain a magnified ‘virtual image’ (final image), which can be seen by eye through the eyepiece. As light passes directly from the source to the eye through the two lenses, the field of vision is brightly illuminated. That is why; it is a bright-field microscope. Parts of a Compound Microscope:

The parts of a compound microscope are of two categories as given below: (i) Mechanical Parts: These are the parts, which support the optical parts and help in their adjustment for focusing the object. The components of mechanical parts are as follows: 1. Base or Metal Stand: The whole microscope rests on this base. Mirror, if present, is fitted to it. 2. Pillars: 2|Page

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It is a pair of elevations on the base, by which the body of the microscope is held to the base 3. Inclination joint: It is a movable joint, through which the body of the microscope is held to the base by the pillars. The body can be bent at this joint into any inclined position, as desired by the observer, for easier observation. In new models, the body is permanently fixed to the base in an inclined position, thus needing no pillar or joint. 4. Curved Arm: It is a curved structure held by the pillars. It holds the stage, body tube, fine adjustment and coarse adjustment. 5. Body Tube: It is usually a vertical tube holding the eyepiece at the top and the revolving nosepiece with the objectives at the bottom. The length of the draw tube is called ‘mechanical tube length’ and is usually 140-180 mm (mostly 160 mm). 6. Draw Tube: It is the upper part of the body tube, slightly narrower, into which the eyepiece is slipped during observation. 7. Coarse Adjustment: It is a knob with rack and pinion mechanism to move the body tube up and down for focusing the object in the visible field. As rotation of the knob through a small angle moves the body tube through a long distance relative to the object, it can perform coarse adjustment. In modern microscopes, it moves the stage up and down and the body tube is fixed to the arm. 8. Fine Adjustment: It is a relatively smaller knob. Its rotation through a large angle can move the body tube only through a small vertical distance. It is used for fine adjustment to get the final clear image. In modern microscopes, fine adjustment is done by moving the stage up and down by the fine adjustment. 9. Stage: It is a horizontal platform projecting from the curved arm. It has a hole at the center, upon which the object to be viewed is placed on a slide. Light from the light source below the stage passes through the object into the objective. 10. Mechanical Stage (Slide Mover): 3|Page

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Mechanical stage consists of two knobs with rack and pinion mechanism. The slide containing the object is clipped to it and moved on the stage in two dimensions by rotating the knobs, so as to focus the required portion of the object. 11. Revolving Nosepiece: It is a rotatable disc at the bottom of the body tube with three or four objectives screwed to it. The objectives have different magnifying powers. Based on the required magnification, the nosepiece is rotated, so that only the objective specified for the required magnification remains in line with the light path.

(ii) Optical Parts: These parts are involved in passing the light through the object and magnifying its size. The components of optical parts include the following: 1. Light Source: Modern microscopes have in-built electric light source in the base. The source is connected to the mains through a regulator, which controls the brightness of the field. But in old models, a mirror is used as the light source. It is fixed to the base by a binnacle, through which it can be rotated, so as to converge light on the object. The mirror is plane on one side and concave on the other. It should be used in the following manner: (a) Condenser Present: Only plane side of the mirror should be used, as the condenser converges the light rays. (b) Condenser Absent: (i) Daylight: Plane or concave (plane is easier) (ii) Small artificial light: High power objective: Plane side Low power objective: Concave side 2. Diaphragm: 4|Page

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If light coming from the light source is brilliant and all the light is allowed to pass to the object through the condenser, the object gets brilliantly illuminated and cannot be visualized properly. Therefore, an iris diaphragm is fixed below the condenser to control the amount of light entering into the condenser.

3. Condenser: The condenser or sub-stage condenser is located between the light source and the stage. It has a series of lenses to converge on the object, light rays coming from the light source. After passing through the object, the light rays enter into the objective. The ‘light condensing’, ‘light converging’ or ‘light gathering’ capacity of a condenser is called ‘numerical aperture of the condenser’. Similarly, the ‘light gathering’ capacity of an objective is called ‘numerical aperture of the objective’. If the condenser converges light in a wide angle, its numerical aperture is greater and vice versa. If the condenser has such numerical aperture that it sends light through the object with an angle sufficiently large to fill the aperture back lens of the objective, the objective shows its highest numerical aperture. Most common condensers have numerical aperture. If the numerical aperture of the condenser is smaller than that of objective, the peripheral portion of the back lens of the objective is illuminated and the image has poor visibility. On the other hand, if numerical aperture of condenser is greater than that of the objective, back lens may receive too much light resulting in a decrease in contrast.

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There are three types of condensers as follows: (a) Abbe condenser (Numerical aperture=1.25): It is extensively used. (b) Variable focus condenser (Numerical aperture =1.25) (c) Achromatic condenser (Numerical aperture =1.40): It has been corrected for both spherical and chromatic aberration and is used in research microscopes and photomicrographs. 4. Objective: It is the most important lens in a microscope. Usually three objectives with different magnifying powers are screwed to the revolving nosepiece. The objectives are: 5|Page

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(a) Low power objective (X 10): It produces ten times magnification of the object. (b) High dry objective (X 40): It gives a magnification of forty times. (c) Oil-immersion objective (X100): It gives a magnification of hundred times, when immersion oil fills the space between the object and the objective. The scanning objective (X4) is optional. The primary magnification (X4, X10, X40 or X100) provided by each objective is engraved on its barrel. The oilimmersion objective has a ring engraved on it towards the tip of the barrel.

Resolving Power of Objective: It is the ability of the objective to resolve each point on the minute object into widely spaced points, so that the points in the image can be seen as distinct and separate from one another, so as to get a clear un-blurred image. Total magnification: The total magnification obtained in a compound microscope is the product of objective magnification and ocular magnification. Mt = Mob X Moc Where, Mt = Total magnification, Mob = Objective magnification and Moc = Ocular magnification

USE OF A MICROSCOPE To achieve high quality microscopical reporting and prevent damage to the expensive optics and moving parts of a microscope, laboratory staff must be trained to use a microscope correctly and to handle it with care. Using a microscope with built-in illumination

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Position the microscope in a shaded part of the room on a secure bench or table that is free from vibration. Do not place it in front of a window where bright daylight will cause unwanted glare. Use a seat of the correct height, i.e. one that will enable the microscope to be used with the back straight. Too high a seat will cause back and neckstrain. If the seat is too high and difficult to adjust, raise the microscope to a convenient height by placing it on a block of wood. Identifying the parts of the microscope Use the manufacturer’s artwork to identify the essential parts of the microscope. The most important parts to identify are:     

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Binocular head and its eyepieces. Revolving nosepiece (objective turret) with 10X, 40X and 100X objectives. On some microscopes a 4X objective may also be fitted. Mechanical stage with slide holder, spring arm, and movement controls. Abbe condenser with iris diaphragm and iris lever. Precentred condenser: A single screw will be seen which is used to remove the condenser should it ever become damaged and need repairing. When the condenser is not one that has been precentred, three centring screws will be seen. Swing-out filter holder with stop for holding it in place. Knob to focus the condenser i.e. move it up and down, positioned on left. Coarse and fine focusing controls on each side. Illuminator with lens (ground glass, light diffusing screen is located below lens). Lamp brightness control. Power ON/OFF switch. Mains cable socket, located at the back of the microscope. Fuse housing.

Setting up the microscope for routine use The following instructions apply to microscopes fitted with an Abbe condenser and illumination system. For microscopes having special condensers and a more complex illumination e.g. illuminator with a field diaphragm, the manufacturer’s instructions must be followed. i. ii.

Turn the rotary lamp brightness control anticlockwise to its lowest setting and then switch on the microscope. Turn up the brightness control to about three quarters of its full power (final adjustment will be made at a later stage).

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iii.

Carefully revolve the nosepiece until the 10Xobjective is located vertically above the stage. iv. Make sure there is no danger of the objective hitting the stage. If this appears likely, turn the coarse focusing control to increase the distance between the stage and objective (in modern microscopes the stage will move downwards whereas in older microscopes the objectives will move upwards). v. Prepare a specimen slide such as a mounted stained thin blood film. A temporary mounted preparation can be made by adding a drop of oil to the lower third of the blood film and covering it with a cover glass. Make sure the underside of the slide is dry, clean, and free of stain marks. vi. Place the specimen slide, cover glass uppermost, on the front of the stage. Gently holding back the spring arm of the mechanical stage, push the slide back into the slide holder and release the arm slowly. The specimen will be held firmly. vii. Important: The spring arm and mechanisms of the mechanical stage can be easily damaged if the surface of the stage or underside of the slide is wet or has oil on it. viii. While looking from the side (not down the eyepieces), turn the coarse focusing control to bring the specimen close to the objective i.e. about 5 mm from the objective. ix. Looking down through the eyepieces, bring the specimen into focus by slowly turning the coarse focusing control in the opposite direction to increase the distance between the specimen and objective. The specimen will come into focus, first as a blurred image and then as a clear image. x. Use the fine focusing control to obtain a sharp image (this will not be the best image because the condenser has yet to be focused and the illumination adjusted). xi. Focus the condenser as follows:  Using the condenser focusing knob located on the left, raise the condenser to its topmost position.  Using the iris lever, open the iris fully.  Check that the filter holder is located against its stop and not out of position and blocking the light.  Looking down the eyepieces and with the specimen in focus, slowly lower the condenser until the mottled image of the ground glass light diffusing screen (located below the lens of the illuminator) is seen in the background.  Slowly raise the condenser until the mottled image of the diffusing screen just disappears (this is usually about 1 mm below the condenser’s topmost position). The condenser is now in focus and should be left in this position. 8|Page

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xii.

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LABORATORY MICROSCOPE

Note: Do not lower or raise the condenser when changing objectives and examining different specimens. The best images will be achieved by adjusting the lamp brightness control and using the iris lever to adjust the condenser aperture (explained later). Check the centring of the condenser unless the microscope is fitted with a precentred condenser (if precentred there will be no centring screws, only a single screw holding the condenser in its mount). To check the centring of a condenser that is not precentred:

Remove the eyepieces and with the eyes positioned close to the eyepiece tubes, close the iris. Note whether the spot of light seen at the bottom of the eyepiece tubes is centrally located. If it is, the condenser is centred correctly and no adjustment is necessary. If the spot of light is off-centre, use the condenser centrifuge screws to adjust the condenser in its mount until the spot of light is centrally placed. Replace the eyepieces.

Note: Once centred correctly, the condenser should remain centred unless it is transported when its centring should again be checked before use. xiii.

Looking down the eyepieces with the specimen in focus, obtain the best possible image by adjusting the condenser aperture and lamp brightness control. For the 10x objective, the condenser will need to be closed about two thirds to provide a good image. Adjust the lamp brightness control to a level which provides good illumination without glare. Important: Use the lamp brightness control, not the iris to reduce the intensity of illumination. If the condenser aperture is closed too much there will be loss of detail (resolution) in the image. If not sure how far to close the condenser aperture, remove the eyepieces and with the specimen in focus, look down the eyepiece tubes and slowly open and close the iris. The condenser aperture is closed by the correct amount when the edge of the iris just comes into view.

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xiv.

LABORATORY MICROSCOPE

Examine the specimen with the 40x objective. Carefully revolve the nosepiece to bring the 40x objective into place. It will locate very close to the specimen. Providing the objectives are parfocal (in focus one with another), only slight focusing with the fine focusing control should be necessary to bring the specimen into sharp focus. Open the condenser iris more and increase the brightness control to obtain a bright clear image.

xv.

Examine the specimen with the 100x oil immersion objective. Revolve the nosepiece to move the 40x objective to one side and before bringing the 100x objective into position, place a drop of immersion oil on the specimen. Carefully locate the 100_ objective. The lens of this objective should just dip into the drop of oil (providing the objectives are parfocal). Use the fine focusing control to focus the specimen. Open the condenser iris fully and increase the illumination to give a bright clear image. Non-drying immersion oil Use a good quality non-drying synthetic immersion oil because this will not dry on a lens should the oil not be removed from the objective after use. Never mix oils. When changing oils, always remove completely the previous oil from an objective before using a new oil otherwise the image will appear blurred.

xvi.

Before removing the specimen from under the oil immersion objective, revolve the nosepiece so that the objective moves to one side. Only then remove the slide from the slide holder.

Important: Moving the objective to one side will not alter its focusing. It will prevent what is the commonest way in which very expensive oil immersion objectives become damaged and need replacing. Scratching of the 100x objective lens can be easily seen by unscrewing the objective and examining the front lens of the objective with a magnifying lens. When used inverted, one of the eyepieces of the microscope can act as a magnifying lens (remove one of the eyepieces and turn it the other way up). Note: To achieve high quality microscopy, prepare specimens correctly using standardized staining techniques and preparations of the correct thickness. Important points when examining specimens

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LABORATORY MICROSCOPE

Before using a microscope, first clean the eyepieces, objectives, condenser lens, stage, and illuminator lens (explained more fully under quality control, care and maintenance of the microscope). Use a cover glass when examining a specimen with the 10x or 40x objective. When using the 100x obj...