Aim: To study the compound microscope.
Requirements: Compound Microscope, permanent slide (or any other specimen).
Theory: The microscope is one of the most commonly used instruments in medical, paramedical, and clinical laboratories. It is used to study Cell Morphology, Histology, Histopathology, and Microbiology. A microscope helps us to see microscopic objects that are too small and invisible to the naked eye.
Description of Compound Microscope:
The compound microscope has the following main parts:
- The supporting system.
- The focusing system.
- The optical or magnifying system.
- The illumination system including: (a) Source of light. (b) Mirror. (c) Condenser.
1. The support system: It is a framework to which various functional units are attached. It consists of the following:
- Base: It is a heavy metallic, ‘U’ shaped or ‘horseshoe-shaped base which supports the microscope on the work table and provides maximum stability.
- Pillars: Two upright pillars project up from the base and are attached to the ‘C’ shaped handle. This allows the microscope to be tilted at a suitable angle for comfortable observation.
- Body tube: It is a 16-17 cm long cylindrical tube fitted at the upper end of the handle which is vertical or at an angle through which light passes via the eyepiece to the observer’s eye visualizing the image.
- The stage: The stage is a square platform with an aperture in its center and fitted to the limb below the objective lenses. When the slide is placed on it, converging rays of light emerging from the condenser pass through the slide and then the objective lens into the body tube. It can be either the fixed stage or the mechanical stage. The fixed stage has two clips that hold the slide in position. The mechanical stage has a calibrated metal frame fitted on the right side of the stage. It has a spring-mounted clip to hold the slide and two screw heads to move the slide from side to side, forward and backward. The Vernier scale is also attached to indicate the degree of movement.
2. The focusing system: The focusing system consists of coarse and fine adjustment and screw heads are used for raising and lowering the body tube for proper focusing of the slide. The coarse adjustment moves the focusing system up or down through a large distance via a rack. The fine adjustment works in the same way which requires several rotations to move the tube over a small distance. It is employed for accurate focusing.
3. The optical or magnifying system: It consists of the body tube, eyepiece, and nosepiece. The body tube is present between the upper end of the objective and the eyepiece. The eyepiece fits on top of the body tube. They can be 5X, 6X, 8X, 10X, or 15X. Each eyepiece has two lenses; the eye lens at the top and the field lens at the bottom. The field lens collects divergent rays and passes through the eye lens to further magnify the image. The nosepiece has two parts; the fixed nosepiece and the revolving nosepiece. The fixed nosepiece holds the revolving nosepiece that carries interchangeable objective lenses. The objective lenses are spring-loaded objectives of different magnifying powers. Different types of objective lenses are low power objective or 10X, high power objective or 45X, oil immersion objectives or 100X, and scanning objective 3X.
Magnification: Magnification is the ability to make small objects seen larger, such as making a microscopic organism visible. The objective lenses magnify the images as stated below:
- Low power objective (10X) = 10 × 10 = 100 times.
- High power objective (45X) = 45 × 10 = 450 times.
- Oil immersion objective (100X) = 100 × 10 = 1000 times.
The oil immersion (100X) objective has a very small aperture and deep focusing position i.e. 1 mm from the slide. The light rays coming from the slide (denser medium) are refracted by a thin layer of air (rarer medium) away from the small aperture of the objective and result in a faint image. If some other medium like cedarwood oil, paraffin, or glycerin having the same refractive index as that of glass is added to the slide, it removes the thin layer of air and forms a continuous medium. This avoids the refraction of light rays and results in a sharp image.
4. The illumination system: The microscope will function only when proper illumination or lightning is provided. The illumination system is to provide uniform, soft and bright illumination. The illumination system consists of:
- Source of light: It may be external (natural day-light, electric lamp, or tube light) or internal (electric in-built light source).
- A condenser: It is a system of lenses fitted as a short cylinder mounted below the stage.
- Mirror: A double-sided mirror with one flat side and the other concave is located below the condenser and can be rotated in all directions. It focuses light rays into a solid cone of light onto the material under study and helps in resolving the image.
- Iris diaphragm: Iris diaphragm is a thin opaque membranous structure fitted within a condenser with a small lever on the side. The lever can adjust the size of the aperture of the diaphragm and allows less or more light to fall on the slide.
- Examine the permanent slide/blood film/specimen first with the naked eye.
- Place the microscope on the working table in an upright position, and raise the body tube approximately 7-8 cm above the stage. Put the slide on the stage and using the mechanical stage, bring the specimen over the central aperture.
- Select the low magnification objective (10X).
- Select and adjust the mirror (plane or concave) so that the light shines on the specimen.
- Adjust the condenser well down, and partly close the diaphragm to cut down excess light.
- Looking from the side, and using the coarse adjustment, brings the body tube down so that the low power lens is about 1 cm above the slide. Look into the eyepiece and gently raise the tube till the slide comes into focus.
- Then choose the area of interest for viewing it under higher magnifications.
- For focusing under high magnification, simply rotate the nosepiece so that the high magnification objective (45X) ‘clicks’ into position. Raise the condenser to the mid position and open the diaphragm to admit enough light. Use fine adjustments as required.
- For focusing on the oil immersion objective (100X), raise the body tube 8-10 cm above the slide. Place a drop of cedarwood oil, paraffin, or glycerin on the slide. Looking from the side brings down the objective till it just enters the oil drop. Use other adjustments as required.
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