BIOLOGY Volume 1 - A Guide to General Biology - 2004

5. CELLS

5.11. Using a Hand Lens and Microscope

5.11.2. Light Microscope

A Light Microscope produces an enlarged image of very small objects using the magnifying power of two convex lenses.

Magnification

Magnification of an object under a microscope is achieved through the combination of the eyepiece lens and the objective lens (Table 5.4).

Class="center">Table 5.4. Microscope Magnification

PARTS OF THE Microscope

Fig. 5.36 shows a microscope with its main structural components labeled. A microscope is a delicate and expensive instrument, so it must be handled with care by following these rules:

1) store the microscope in a cabinet (or under a dust cover) to protect it from dust;

2) when removing the microscope from its storage, hold it by the arm with one hand and support the base with the other; place it down gently, avoiding any sudden jolts or impacts;

3) the lenses must be kept clean by wiping them with a suitable piece of cloth (see below).

Fig. 5.36. Light microscope.

Care of Lenses and Glassware

Dirt (dust, grit, grease, etc.) is one of the main challenges when working with a microscope, so the lenses and slides must be kept clean at all times. You can wipe them before starting your work, but if you notice dirt while viewing, try to locate its source. For example, while looking through the microscope, move the microscope slide slightly or rotate the eyepiece lens. If the smudge remains stationary in both cases, the dirt is likely on the objective lens, the mirror, or the side of the condenser lens facing the light source.

First, try blowing away any loose particles; if the dirt persists, gently brush it off with a fine brush or a piece of cloth. Breathe on the Glass or lens to moisten the surface slightly, then wipe it dry with a soft cloth. (Always use specialized lens paper or lint-free cloth that contains no wood fibers, as these can scratch the glass. Standard fabrics are not suitable for this purpose. A soft, washed cotton cloth or lens tissue works well.)

If necessary, the eyepiece lens can be removed for cleaning, and the objective lenses can be unscrewed. After cleaning, the lenses must be replaced immediately to prevent dust from entering the microscope.

Always follow these guidelines:

1) hold microscope slides by their edges and avoid touching the coverslip with your fingers;

2) never Touch the lenses with your fingers;

3) keep the stage clean;

4) cover the microscope and slides when they are not in use.

Setting up the microscope for low-power observation.

1. Place the microscope on a workbench and sit comfortably. Arrange all the necessary equipment on the table within easy reach. The specimen on the microscope stage must be adequately illuminated. This can be achieved using a specialized illuminator, natural light from a window, or a desk lamp. In the latter two cases, the concave surface of the mirror located beneath the stage is used. Direct the light through the aperture in the stage using the mirror. If a suitable condenser is available, use the flat surface of the mirror to direct light through it. The field of view should be evenly illuminated.

2. Using the coarse adjustment knob, raise the microscope body tube and rotate the nosepiece until the low-power objective (x10 or 16 mm) clicks into place. The magnification power of the lens is typically indicated on its barrel.

3. Place the slide you wish to examine on the stage so that the specimen, covered by the coverslip, is positioned directly over the center of the stage aperture, allowing light to pass through it.

4. Looking at the stage and the slide from the side, lower the tube using the coarse adjustment knob until the low-power objective is approximately 5 mm away from the specimen.

5. Looking through the microscope, slowly raise the tube using the coarse adjustment knob until the specimen comes into focus.

6. Always focus the microscope by moving the tube upward rather than downward. Otherwise, you may easily miss the focal point and damage the slide.

7. Keep both eyes open, alternating between them.

Difficulty in viewing a specimen is usually caused either by dust or debris on the coverslip coming into focus, or by an incorrect objective lens position (being too far from the specimen).

Moving the Specimen

Notice in which direction the specimen shifts when you move the glass slide left and right, toward and away from you. This will help you locate specific points on the slide or track a moving object. A graduated mechanical stage allows you to repeatedly return to a point of interest on the slide.

Setting up the microscope for high-power observation

1. When working with a high-power objective, artificial lighting is required to ensure sufficient illumination. Use a desk lamp or a specialized microscope illuminator with a frosted bulb. When using an incandescent lamp, place a sheet of paper between the lamp and the microscope. Rotate the mirror so that its flat surface faces upward, reflecting the light into the microscope.

2. Focus the condenser without removing the slide from the stage. Raise the condenser (Fig. 5.36) so that the distance between it and the stage does not exceed 5 mm. Looking through the microscope, turn the coarse adjustment knob until the specimen is in focus. Next, adjust the condenser focus until the image of the lamp filament is superimposed precisely on the specimen. Move the condenser slightly out of focus so that the lamp image disappears. Illumination should now be optimal. An iris Diaphragm is built into the condenser to regulate the size of the aperture through which light passes. This aperture should be opened as wide as possible to achieve maximum image clarity.

3. Rotate the nosepiece until the high-power objective (x40 or 4 mm) clicks into place. If the focus was already set at low power, rotating the nosepiece will automatically bring the high-power objective close to the focal plane. Always perform final focusing by moving the objective upward using the fine adjustment knob.

4. If the image cannot be focused when switching to the high-power objective, proceed as follows: viewing the stage from the side, lower the microscope tube until the lens is nearly touching the slide. Watch the reflection of the objective lens on the slide and adjust until the lens almost touches its own reflection.

5. Looking through the microscope and turning the fine adjustment knob, slowly raise the objective until the image comes into focus.

Oil Immersion

To achieve higher magnification than is possible with a standard high-power objective (x400), an oil-immersion lens must be used. The light-gathering capacity of the lens is significantly enhanced by placing a liquid between the objective lens and the coverslip. This liquid must have the same refractive index as the glass lens itself. Therefore, cedarwood oil is commonly used as the immersion medium.

1. Place the slide on the stage and focus the image just as you would for standard high-power observation. Rotate the nosepiece to switch from the high-power objective to the oil-immersion lens.

2. Place a drop of cedarwood oil directly onto the coverslip directly above the area of the specimen you wish to study.

3. Focus the image first under low power, then rotate the nosepiece to click the oil-immersion objective into place so that its tip dips into the drop of oil.

4. Looking through the microscope, very carefully bring the lens into focus using the fine adjustment knob. Remember that the working focal distance of the lens is only about 1 mm from The surface of the coverslip.

5. When finished, gently wipe the oil off the lens with a soft lens tissue.

Measuring Microscopic Objects (Micrometry)

The need to determine the exact dimensions of structures studied under a microscope is entirely obvious. The measurement of microscopic objects is called micrometry. Special scales, or micrometers, are used for this purpose. One such scale (the ocular micrometer or eyepiece micrometer) is inserted into the eyepiece, while the other (the stage micrometer) is placed on the microscope stage (Fig. 5.37). Both scales are uniform, but the absolute value of a single division is known only for the stage micrometer.

Fig. 5.37. A. Ocular micrometer. B. Typical scale of a stage micrometer. Total length — 1 mm.

Handle micrometers with care, making sure not to leave fingerprints on them. Hold them only by the edges to avoid accidental scratching.

Before measuring any object with an ocular micrometer, you must determine the calibration value (division value) of the ocular micrometer at each magnification. In other words, the ocular micrometer needs to be calibrated. To do this, place the stage micrometer on the stage instead of the specimen and count how many of its divisions correspond to a known number of ocular micrometer divisions at the given magnification. Follow these steps:

1. Unscrew the top lens of the eyepiece and insert the micrometer into the eyepiece, ensuring that the scale faces upward. The micrometer will rest horizontally, supported by the internal ledge inside the eyepiece. Screw the lens back into place.

2. Place the stage micrometer on the microscope stage. Light must pass through the scale so that it is clearly visible through the microscope.

3. Rotate the nosepiece to click the low-power objective into position, and slowly raise it using the coarse adjustment knob until the scale comes into focus.

4. Rotate the eyepiece lens to align the ocular micrometer scale parallel to the stage micrometer scale, and then shift the latter so that the readings of both scales can be compared.

5. Determine as accurately as possible how many stage micrometer divisions correspond to a known number of ocular micrometer divisions. (The more divisions you use, the greater the accuracy will be.)

6. The division value of the stage micrometer may be 0.1 mm or 0.01 mm (this value is indicated on it). Knowing this, you can calculate the absolute value of a single ocular micrometer division for the given magnification.

7. Repeat this Procedure for any other objective lenses you intend to use (and, if necessary, for any other eyepiece lenses as well). The calibration of each ocular micrometer is strictly specific to that particular combination of lenses and microscope.

8. Remove the stage micrometer from the stage and place your specimen in its place. You can now measure any part of the specimen in ocular micrometer divisions. By rotating the eyepiece lens, align the ocular micrometer scale parallel to the feature of the specimen you wish to measure.

Converting ocular micrometer divisions into absolute values is easier using a graph. To do this, plot the number of ocular micrometer divisions (e.g., up to 100) on the y-axis, and the dimensions in millimeters on the x-axis (Fig. 5.38). For each magnification, find the two points corresponding to 1 and 100 Divisions of the ocular micrometer, and connect them with a straight line. Plot these graphs for each magnification within the same coordinate system, using appropriate scales for the x-axis. You can now determine the size in millimeters for any number of ocular micrometer divisions at any magnification. It is always convenient to keep such graphs close at hand alongside the microscope.

Fig. 5.38. Graph for converting ocular micrometer divisions into micrometers.