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USE OF COMPOUND MICROSCOPE © Institute of Lifelong Learning, University of Delhi 1 USE OF COMPOUND MICROSCOPE LIFE SCIENCES (LAB MANUAL) © Institute of Lifelong Learning, University of Delhi 2 LIFE SCIENCES (LAB MANUAL) USE OF COMPOUND MICROSCOPE Aim To learn the use of a light microscope in the laboratory. Learning Objectives Life Sciences laboratories are usually concerned with organisms so small that they cannot be seen distinctly with the naked eyes. In such laboratories, Microscope is of crucial importance and is commonly used in the lab practical. By this exercise we will be able to know various parts of this instrument and also to learn the working of the compound microscope with best possible results. Materials / Requirements Equipment: A Light Microscope Material: Any simple permanent slide may be of animal/plant tissue. Theoretical aspects of the experiment The microscope is employed to see fine details which are not visible to the naked eye. The primary function of the common lab microscope is not to obtain a very high magnification but to yield an image in which small details are optically resolved. The most common type of microscope found in laboratories is the LIGHT MICROSCOPE also called as COMPOUND MICROSCOPE which is an optical microscope capable of magnifying the apparent size of an object. Visible light is projected through the specimen such as a squamous epithelium mounted on a slide and is kept on the stage of the microscope. Glass lenses, the eye piece and the objective then enlarges the image and project it to a human eye. The two important factors in microscopy are the magnification and the resolving power. Magnification means an increase in an apparent size of the object as compared to its actual size. Resolving power or resolution is the smallest distance between two point-like objects which can just be recognized as separate. It is achieved if the visual angle between the object details themselves, or in their magnified image, is large enough so that light rays from either point will impinge upon different receptor elements in the retina. Each optical instrument such as microscope, telescope has a limit to its resolving power. For example, human eye can resolve points that are as close together as 1/10 millimeter (mm). The resolving power of light microscope is about 0.2 micrometer (µm). Thus, the principle of working of a compound microscope is that when a ray of light passes from one medium to another, REFRACTION occurs. This means that the ray is bent at the interface. The REFRACTIVE INDEX is a measure of how greatly a substance slows the velocity of light, and the direction and magnitude of bending is determined by the refractive index of the two media forming the interface. © Institute of Lifelong Learning, University of Delhi 3 LIFE SCIENCES (LAB MANUAL) USE OF COMPOUND MICROSCOPE The most important part of the microscope is the objective, which produces a clear image, not just magnifies one. The resolution and magnification are extremely important in viewing an object in the microscope. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. The minimum distance (d) between two objects that reveals them as separate entities is given by the following equation: d 0.5 sin Lambda () is wavelength of light used to illuminate the specimens nSin is the numerical operator As d becomes smaller the resolution increases. To achieve higher resolution, increase refractor index using immersion oil which is a colorless liquid with same refractive index as glass. If air is replaced with immersion oil, many light rays that did not enter the objective due to reflection and refraction at the surface of the objective lens and slide will now do so. Preparation for the experiment A compound microscope with its component parts A permanent slide/temporary mount of a animal/plant tissue © Institute of Lifelong Learning, University of Delhi 4 LIFE SCIENCES (LAB MANUAL) USE OF COMPOUND MICROSCOPE The microscope consists of the following parts as shown in the Figure: 1. 2. 3. 4. Study metal body. Stand composed of BASE and an ARM to which remaining parts are attached. A light source, either a mirror or an electric illuminator located at the base. Two focusing knobs the fine and coarse adjustment knobs. These are located on the arm and can move either stage or the nosepiece to focus the image. 5. The stage is positioned a out halfway up the arm. 6. Microscope slides are held by simple slide clips. These clips are fixed on the stage. 7. A mechanical stage allows the operator to move a slide around smoothly during viewing by use of stage control knobs. 8. The sub stage condenser is mounted within or beneath the stage and focuses a cone of light on the slide. 9. The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more eyepieces or oculars are attached. 10. The nose piece hold three to four objectives with lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly. © Institute of Lifelong Learning, University of Delhi 5 USE OF COMPOUND MICROSCOPE LIFE SCIENCES (LAB MANUAL) Procedure of using a microscope 1. 2. 3. 4. 5. 6. 7. 8. 9. Place the microscope on a stable place on a laboratory bench. Place a stool behind the laboratory table for sitting to look into the microscope. The stool should have a convenient height to let you see through eye piece without bending. Place microscope near the window if daylight is used for illumination. Provide illumination from any of the three sources – build in lamp, external lamp or sunlight. Use the concern side of the mirror. Use the plane side of the mirror if there is condenser. Direct the path of light to pass through the hole of the stage with maximum intensity while setting the mirror. Put the slide between the clips provided on the stage. Revolve the nosepiece and align the low power objective (10X) to examine the object on the slide. Adjust illumination to improve the clarity of the object on the slide. Put one hand on the focusing knob (coarse or fine) and another on the screw to move the stage. The low power objective is commonly used to screen the field of view. Bring the objective of interest in the centre. Switch to high power (40X) and increase illumination as needed. Repeat the process of focusing. After screening under the low power and examining under high power, if desired and required, oil-immersion objective is used for obtaining greater details of the object. Sources of error and precautions Care is required at each stage in handling the microscope and also during focusing of slide. Always sit straight. The stool used for sitting to look into the microscope should have a convenient height to let you see through eye piece without bending. Always focus the slide first under low power. Use lens paper/tissue paper to clean the mirror, eyepiece and objectives. Never use your fingers to clean them. Carefully use the fine adjustment while focusing the slide under high power. Clean the stage, lenses etc. after finishing the work with the microscope, never leave it dirty. Always hold the microscope with both hands, putting one hand below the base to prevent falling of mirror while carrying it from one place to another. Observations Magnification is the magnifying capability of a compound microscope. It is the product of the individual magnifying powers of the OCULAR Lens (the lens nearest to eye) and objective lens (lens nearest to specimen). A typical microscope used in an undergraduate laboratory has objective lens of 10s, 40s and 100s and an ocular lens of 10x, thus capable of magnifying the image of specimens 100, 400 and 1,000 times respectively. One has to observe the specimen present on the slide at various magnifications. © Institute of Lifelong Learning, University of Delhi 6 LIFE SCIENCES (LAB MANUAL) USE OF COMPOUND MICROSCOPE Results The structure of a tissue is seen clearly under low and high powers. Discussion/Interpretation The working of the light microscope is to be shown with the help of a ray diagram. Questions 1. 2. 3. 4. What are compound microscopes? What are the different microscope magnifications and what do they mean? Is it superior to have one eyepiece or two? What are the several problems associated with light microscopy? © Institute of Lifelong Learning, University of Delhi 7