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The Microscope The microscope plays a very important role in laboratory for the tissues and organisms which

are too small to be seen clearly with the naked eye. With the aid of the microscope, however, these objects may be magnified so that an entirely new world of life is opened to our vision. Such an instrument, is of necessity, very delicate and built to high precision, hence it must be handled and used with care and understanding in order to obtain the desired results and to avoid serious damage to the microscope. Please keep these facts in your mind before you use your microscope. Even though you have used a microscope before it is quite likely that there are many points about its use which you have forgotten, or never knew, and you should make the same careful study about its use as the others who have never touch a microscope before. In order to understand the proper use of a microscope we must first learn the parts that make it up and function of each. The drawing of a microscope giving here will serve a guide to the identification of the parts. Learn to identify all of these parts and to know them by names in the directions to follow and you cannot understand the directions if you do not know the names of the parts. There are several points should also be noticed: The usual class microscope has eyepieces/oculars magnifying x 10, and an objective nosepiece carrying x 4, x 10, x 95 (or x 100) (oil immersion) lenses. Normally the three lower-power lenses are mounted on the nosepieces, whilst the oil immersion objective may be mounted or kept separate. Every time it is used, the microscope should be set up to the best optical advantage. Keep in mind the limit to resolution. The section has some thickness, so that the fine-focusing adjustment should be used continually during observation to bring out fine detail, e.g., cilia on cells. Essentially, though, we are getting a two-dimensional picture from an originally three-dimensional piece of material. For what the structure looked like in the third dimension, you can try to reconstruct mentally what is going in the missing dimension, and look up views of the structure in scanning electron microscopy. Artifacts (appearances not due to the original nature of the material as obtained from the body) can arise at all stages in the treatment of the section. Gross examples arise from: o clumsy excision from the body; o poor or inappropriate fixation; o shrinkage and worse, uneven shrinkage leading to artificial spaces and distorted relations; o cutting scores or chatters from a bad microtome knife; o section not flat on the slide; o water, dirt or bubbles on in the section; o dirt on the microscope lenses; o patchy or faded staining, unbalanced staining when more than one stain has been applied;

o precipitate from fixative or stain; o tears and folds in the section. Setting up the microscope o Adjustment of light by using artificial or natural light. o Light intensity can be increased by bring the lamp nearer to the mirror, if the lamp is not built-in. o If the condenser is in use (nearly always), use the plane side of the mirror to reflect light accurately up the optical axis of the microscope. o Raise the condenser to very near the underside of the stage, and open the iris diaphragm. o Place a clean, stained slide on the stage and using the coarse and fine adjustment head bring it into focus. o The iris diaphragm should now be closed just to the point where glare is eliminated. Further closure will make the field too dark and reduce resolving power. o The microscope is now set up for use, but the requirements change for each objective. Higher power objectives require more light, thus the iris will need to be opened and perhaps the lamp brought nearer to the mirror and condenser re-fused. o Note that the objective lenses are of different lengths, and they are not always parfocal. Be careful when switching in a higher power lens that it does not hit the slide because of its greater length. Clean the lenses only with lens paper. About the use of the microscope: About eyeglasses. Many students wear glasses and may wonder whether they should keep then on or take them off when viewing objects through the microscope. It the trouble is due to simple near- sightedness or far-sightedness there is no need to wear glasses. They may be worn, but a slight change in focus will be necessary. If the student has astigmatism, it is desirable to wear the glasses while using the microscope since the microscope lenses will not correct for astigmatism. In case using monocular Keep both eyes open while using the microscope. the beginning student may find it tiring to hold one eye closed while looking through the eyepiece with the other. Sometimes one may be observed holding his hand over one eye to relieve this strain. It is possible to see clearly by keep both eyes open. Through practice you soon learn to ignore that which is seen by the eye not over the eyepiece. Never try to find an object under high power if you cannot find it under low power. Often students will be observed searching vainly for an object under high power. When asked what the trouble is they will reply, " Well, I couldn't find it under low power, so I though I would try high power." High power is much more difficult to use than low power, then rest assured you will not find it under high power. When preparing to put the microscope away for the day, always leave it with the low power under the tube and with the diaphragm wide open. There is danger that the high

power lenses may be run down through the open in the stage and damage the condenser or the iris diaphragm is left partially closed. this cannot happen if the lower power is under the tube and the diaphragm is wide open. High power should never be used with a cover glass.

Procedure : Operating procedure: Place the Microscope in an upright position at a convenient height for the operation on a rigid table. Make sure all the exposed Optical surfaces are free of dirt. Plug the power cord into a Grounded outlet. Bring the light intensity regulator to lowest level and switch on the power. Adjust the observation head to convenient working position. Rotate the nose piece until the lowest power objective is in Viewing position. The lower the power of objective, the greater the area of the specimen surface included in the field of view. Lower power objectives also have a much greater depth of focus are generally used for initial focussing and viewing. Take down the stage to a fairly low position with the help of coarse focus knob. Make sure that the stage surface is free of dirt, grit or any other material that will interfere with the movement of the specimen slide and the stage between the stage fingers. Position the specimen area of slide (Cover glass upside) over the centre of stage aperture. Use the stage control knobs to move specimen slide to the desired position. Looking through the observation head, raise the stage by adjusting the coarse focus knob until an image appears. Focus as Sharply as possible with coarse focus knob. Adjust the fine focus to sharpen the image in the centre of the field of view. Looking through the observation head, raise the stage by adjusting the coarse focus knob until an image appears. Focus as sharply as possible image. The clarity of the image depends upon the size of aperture. As the aperture becomes smaller, the contrast and depth of focus increases but the resolving power decreases. The clearest image is produced by combination of these three factors. Examine the specimen. When you find a feature you wish to observe at a higher magnification. Move the slide so that the feature gets centre in the field of view. Use the fine focus knob to sharpen the image. When using objective of higher numerical aperture ( N.A.) proper focussing of the abbe condenser is important. Focus the abbe condenser by racking the condenser movement knob up and down so that the field is evenly illuminated. This microscope has a sliding potentiometer coupled with electronic Circuit for regulating the light intensity slide the potentiometer from low to high as you go from low magnification to high magnification Objectives for obtaining best light in the field of view. The procedure for examining a specimen using the oil immersion objectives is as follows. Rotate the nose piece so that the lower power objective is in light path.

Place one drop of immersion oil ( Cedar wood oil ) on the lighted area of the specimen slide. Dust or air bubbles in the oil can destroy the image. If the bubbles are trapped between the objective lens and the slide, clean off the oil and start again. Rotate the nose piece so that 100X oil immersion objective is in the light path. With your eye at the level of the stage, use coarse focus knob to raise the stage with the specimen cover glass. When you see a flash of light at this location, the objective lens has made contact with the immersion oil and the microscope can be focussed using the fine focus knob. Each time you finish using the oil immersion objective, wipe off all traces of oil from the objective and the specimen cover glass with a clean soft cloth.

Fine adjustment of Binocular head. Rotate the Binocular head to bring it to a convenient position. Adjust the interpupilary distance by bringing the eyepiece tubes closer or apart till you see one fused image. If the image from both occulars does not fuse, you are required to do Dioptric adjustment on the occulars as below. Bring 10X objective in position and focus the slide in the right eye with coarse and fine focus knob keeping the left eye closed. Close the right eye and seeing through the left eye, focus the left Ocular up and down by rotating the focusing sleeve till the image is in sharp focus. Look through both the eyes if interpupilary adjustment required. After viewing, the results should be recorded in the respective report format Cleaning of Microscope After use clean the microscope to moisture and dust free. Wipe the lens surface and objective with a cleaning soft cloth. Use Xylol sparingly to remove oil from the objective. Keep the Microscope in dust & moisture free wooden box. Enter the cleaning details in the log book. Frequency of cleaning: Once in a month. Calibration of Microscope Remove the eye piece and insert the ocular Micrometer. The graduations in the ocular micrometer can be seen under sharp illumination. The lines and distances will remain unchanged under different Objectives. The stage micrometer is mounted on the microscopic field under a sharp focus. This is done first with low power objectives and then under high Power objectives. The scale of ocular micrometer and stage micrometer is adjusted in such a way that the lines of one should superimpose on the lines of other at one end of the microscopic field. The number of divisions are counted lying in between two coinciding divisions of both ocular and stage micrometer. Since we know the value of each division on stage micrometer the Value of division on the ocular can be calculated. Frequency: Once in a month.