PRACTICAL MANUAL OF HISTOLOGY, CYTOLOGY AND EMBRYOLOGY - 2016

Chapter 1. HISTOLOGICAL TECHNIQUE

Structure of the microscope and rules for its operation

Cell/15.html">Microscopy is the primary method for investigating histological specimens.

A Microscope is an optical instrument that provides magnified images of BIOLOGICAL OBJECTS AND reveals structural details that are invisible to the naked eye.

A microscope consists of optical and mechanical components. The optical components include objectives, eyepieces, a mirror, and a condenser with an iris Diaphragm; the mechanical components comprise the base, limb (arm), body tube, revolving nosepiece (turret), stage, coarse and fine adjustment knobs, and condenser positioning controls.

Optical Components of the microscope

Under a microscope, an object is typically observed in transmitted light through an objective and an eyepiece. The light path in a compound microscope is illustrated in Fig. 2. Light rays from the source (1) pass through the condenser (2) and the specimen (3) to the lenses of the objective (4) and eyepiece (5), and further to the observer's eye, forming an intermediate image (6) and a final image (7). Both the objective and the eyepiece consist of a system of lenses secured in metal mounts.

Microscope lenses have The ability to magnify images. Magnification is the property of diverging the light rays emerging from the objective. It is defined as The ratio of the linear dimensions of the image to the linear dimensions of the object. Magnification depends on lens curvature: the greater the curvature, the higher the magnification.

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Fig. 2. Schematic diagram of a Light Microscope.

Accordingly, the greater the curvature, the smaller the diameter and the shorter the focal length. The total magnification of a microscope (the product of the objective and eyepiece magnifications) is approximately 1,500x for a light microscope.

Lenses also ensure image clarity, which depends on the degree of primary optical aberrations—namely, spherical and chromatic aberrations.

Spherical aberration is the stronger refraction of light rays passing through the peripheral zones of a lens compared to those passing through its central region. Spherical aberration causes structural details in the image to become blurred. This aberration can be reduced by diaphragming.

Chromatic aberration is the dispersion of white light into a spectrum by a lens due to the uneven refraction of rays with different wavelengths. This results in color distortion and reduced image clarity. Chromatic aberration can be eliminated or minimized by manufacturing lenses from Different types of optical Glass with varying refractive indices.

Lenses are also characterized by resolving power—the shortest distance between two points of an object at which they can still be distinguished as separate entities. Resolving power (d) is determined by the formula , where λ is the wavelength of light used to observe the object; n is the refractive index of the medium between the object and the objective; and α is the angle between the optical axis of the objective and the most steeply inclined ray entering the objective.

The value $n \cdot \sin \alpha$ is an important optical characteristic known as the numerical aperture of the objective. The resolving power of a light microscope is approximately 0.2 µm, which is close to the theoretical limit.

The objective (1) is the primary optical component of the microscope (Fig. 3). It consists of a system of lenses in a metal mount that magnify the specimen's image. A distinction is made between the front lens, which is closest to the object and forms the primary image, and the upper lens, which is closest to THE EYE AND corrects the aberrations of the front lens.

The MBD-1 biological microscope features three objectives: low-power, high-power, and oil immersion. An objective is characterized by its inherent magnification, numerical aperture, focal length, and other constants. Typically, the first two characteristics are engraved on the objective housing: 8x and 0.20 for the low-power objective; 40x and 0.65 for the high-power objective; and 90x and 1.25 for the oil immersion objective. The low-power objective also differs from the high-power one by having a shorter metal barrel and a larger front lens diameter. The objectives are housed at the lower end of the body tube (2) in a revolving nosepiece (3) (Fig. 3).

The eyepiece is the second most important optical component of the microscope, located at the upper end of the body tube (4). It is used to view the image formed by the objective. A simple eyepiece (Huygens type) consists of two lenses in a metal mount, with a fixed circular diaphragm positioned between them. The upper lens is called the eye lens. Its mount bears the magnification value—such as 7x, 10x, 15x, etc. The lower lens is called the field lens. To calculate the total magnification of the microscope, multiply the objective magnification by the eyepiece magnification.

The mirror (5) directs light rays toward the condenser and features both flat and concave surfaces. It is mounted at the lower part of the stand and can rotate on its axis. The mirror collects light from the illuminator and directs it upward through the condenser onto the specimen. The concave surface produces a more concentrated beam of light.

Fig. 3. Structure OF THE microscope

When a condenser is used, the concave surface of the mirror is employed for low magnification and the flat surface for high magnification; the reverse applies when no condenser is used.

The condenser (2) collects light rays and focuses them onto the specimen, thereby ensuring adequate and even illumination. It consists of two lenses enclosed in a shared mount, secured by a dedicated condenser adjustment screw. The collar (mount) is connected to an iris diaphragm located beneath the condenser. The aperture of the diaphragm is adjusted (opened or closed) by a lever situated on the side.

beneath it. Even lower is the filter holder ring. Typically, two light filters come with the microscope: a colorless frosted filter and a frosted blue filter, which adjusts the color Temperature of the electric light source to closely mimic daylight.

The condenser, iris diaphragm, and light filter can be raised or lowered by turning the adjustment knob located next to them on the right side (6,a).

Mechanical Components of the Microscope

The microscope stand consists of the base, the limb (Column), the body tube, the revolving nosepiece (turret), and both the coarse and fine adjustment knobs.

The base, or FOOT of the stand (7), has a horseshoe or rectangular shape to ensure the microscope remains stable.

The limb (column of the stand) (8) connects at the lower end to the fine mechanism housing and bears the coarse adjustment knob (9) and, positioned just in front of it, the fine adjustment knob (10), as well as the condenser adjustment knob.

At the top, the column terminates in a HEAD (11) shaped like an obliquely truncated cylinder. The revolving nosepiece (3) with slots for objective lenses is movably attached to the underside of the head.

Rotating the nosepiece by hand allows the user to switch between objective lenses needed for examining the specimen slide. At the top, the head features a large threaded socket where a photographic or binocular head (with two tubes) can be inserted if necessary. These attachments are secured in the socket by a set screw located on the side of the limb head.

The column can also serve as a handle when carrying the microscope.

The body tube (2) is designed to hold the eyepiece. In modern microscopes, the body tube is inclined at an angle to the stage. Upward and downward movement is achieved by turning two knobs: the coarse and fine adjustment screws.

The coarse adjustment knob, or rack and pinion (9), is used for rapid, rough positioning of the body tube until a sharp image of the studied object is obtained. The knob features two wheel-like handles on both sides of the microscope and can be rotated clockwise or counterclockwise. The coarse adjustment knob is positioned low enough so that users do not need to lift their HAND FROM THE workbench while operating it.

The fine adjustment knob (10) serves for subsequent, more

precise, and sharp focus adjustments. It moves the body tube by a microscopic distance, which is why it must not and should not be forced or turned too far. One full Rotation of the knob corresponds to a 0.1 mm movement of the body tube.

Turning either the coarse or fine adjustment knobs away from the user (i.e., clockwise) lowers the lenses toward the specimen. Turning them toward the user (i.e., counterclockwise) raises the body tube, moving the lenses away from the specimen.

The mechanical stage (12) is designed to hold the microscope slide. In the center of the stage is a circular aperture through which light rays from the mirror pass to illuminate the specimen from below. The stage contains two small holes where metal spring clips (stage clips) are inserted to securely hold the slide in place.

Stages can be either fixed (square) or mechanical (circular). The latter can be moved using two mechanical stage knobs located on the right and left sides of the stage. Just behind the right-hand knob is another screw: the stage rotation lock. When tightened, the mechanical stage is locked in place.

Rules for Microscope Handling

1. Carry the microscope by holding the column with one hand while supporting the base with the other.

2. Place the microscope on your workbench so that the column faces you and the mirror points toward the light source. Place your drawing notebook on the right side.

3. Check the revolving nosepiece, select the low-power objective (8x), and use the coarse adjustment knob to lower its bottom edge to within 1 cm of the stage.

4. Set up the illumination by adjusting the mirror until the entire field of view is evenly and brightly lit. Check the illumination by looking through the eyepiece with your left eye.

5. Place the microscope slide on the stage with the coverslip facing up. The section must be positioned directly over the stage aperture.

6. Turn the coarse adjustment knob to find a clear image of the organ or tissue structures in the section, select an area of interest, and center it in the microscope's field of view. Afterward, secure the slide with the stage clips.

7. When switching to a high-power objective, you must: raise the microscope body tube by turning the coarse adjustment knob toward you, smoothly

rotate the nosepiece to click the high-power objective (40x) into place. Next, viewing the microscope from the side, turn the coarse adjustment knob away from you until the front lens of the objective is as close as possible to the coverslip. Then, looking through the eyepiece, very slowly turn the coarse adjustment knob toward you until a roughly focused image appears. Follow this by slowly turning the fine adjustment knob until a sharp image is achieved. Always remember that careless handling can crush the microscope slide.

8. When moving to examine another area of the slide, return to low magnification and repeat steps 6 and 7.

9. Examine and study the specimen under high magnification and proceed to make a drawing of it.

10. Upon completion of work with the microscope, set the low-magnification objective lens in place.