CARE AND USE OF THE MICROSCOPE

 

After completing this investigation, you should be able to:

            1. Identify the various parts and associated functions of your microscope.

            2. Know how to properly adjust the light sources to achieve optimum illumination.

            3. Take advantage of the parfocal nature of the objectives.

            4. Understand the relationship between magnification and size of the field you are viewing.

            5. Demonstrate your ability to find and focus on an image at all powers.

 

Introduction

 

The bright-field compound microscope is probably the most important instrument you will use in any biology course. Objects such as bacteria, mold spores, plant cells, or animal tissues, too small to be seen with the unaided eye will become highly magnified, allowing you to view minute details.

 

Logic would suggest that the more we magnify the image of a cell, the more detail we can see. Unfortunately, this is not true. Light rays passing through curved surfaces are bent. This bending of light produces an enlarged image. But the best research microscopes can currently magnify only 1500 to 2500 diameters. The limiting factor is the nature of light. Of equal importance with magnification is resolving power. Resolving power or resolution is the ability to distinguish separate images of objects that fall very close together on the retina of the eye. Since the human eye cannot ordinarily resolve objects separated by less than 0.1 mm, a microscope can extend the resolving power of the eye about 400 times. Thus, a microscope provides magnification and permits us to see objects as separate images that are close together and would appear as a single object to our eyes.

 

Parts of the Compound Microscope (relate the information to Figure x-1)

 

 1. The microscope rests upon the table on its base or foot.

 

   2. Attached to the foot is the upright part known as the arm or pillar of the microscope.

       When carrying the microscope grasp it firmly by the arm. DO NOT TIP IT. If it is too

       heavy, support the microscope by holding one hand under the base.

 

   3. The stage is the platform upon which the slide to be studied rests. Our microscopes have

       mechanical stages which enable you to easily control the movement of the slide you are

       observing. Below or next to the stage, usually on the right hand side, are two milled knobs.

       They control the movement of the mechanical stage. The inner knob will move the

       stage toward or away from you. The outer knob gives a right-left motion to the slide holder.

       To place a slide on the stage, pull back  the lever arm that protrudes from the left side of the

       mechanical stage. This opens the slide holder and allows you to insert the slide into the notched

       area. Release the lever carefully to avoid damage to the slide.

 

   4. The coarse adjustment is the larger milled wheel located near the base of the

       microscope, on each side. This moves the stage up and down and enables you to get an

       approximate focus on the study object. About 90 –95% of your focusing is done with the coarse

       adjustment.

 

   5. The fine adjustment is the smaller milled wheel at each side of the base of the

       microscope. This adjustment moves the stage up and down small distances to bring objects into

       sharp focus. You should be able to use this adjustment to gain knowledge about the relative

       position of the parts of the object you are observing. Focusing up and down will bring new

       parts into clear focus, and tell you that these parts are above or below the parts on which you

       had previously focused. Remember that even though small, objects you are observing are three-

       dimensional. Thus, they require frequent readjustments of focus. This will aid in development

       of a clearer concept of the structural relationships between the parts of the object being viewed. If,

       when attempting to use the fine adjustment, you find that the wheel has been turned as far as it will

       go in one direction, turn the coarse adjustment so that the lenses are farther from the object

       being viewed. Focus again with the coarse adjustment and then turn the fine adjustment. If you

       have correctly  focused with the coarse adjustment, you will not need to turn the fine

       adjustment more than one half of a turn.

 

   6. The revolving nosepiece is at the lower end of the body tube. It normally holds two to

       four objectives. Turn the coarse adjustment so that there is room for the longest adjustment

       without it touching the stage. Now turn the nosepiece and feel the "click" as each objective

       goes into place.

 

Some microscopes have a very short objective which magnifies about 4 times. This is sometimes called the scanning objective and can be used to get a wide view of the field of study. The next shortest objective has a large opening in it and a large lens. This objective is called the low power objective and magnifies about 10 times. In some models of microscopes the number 16 is inscribed in this objective. This indicates that the objective is 16 mm above the object when the object is in focus. The high-dry objective is longer than the 10 power objective and is identified by the number 40 or 45 inscribed on it. It should magnify about 40-45 times.

 

The microscopes in many laboratories, ours included, have an oil immersion objective. Note the size of the aperture (opening) in this lens. To use this lens place a drop of immersion oil on the slide or cover slip. Place the oil exactly where the light is coming through the opening in the stage and onto the slide. The lens is then carefully moved into the oil forming a continuous column from the slide to the objective lens. Light is not refracted upon passing between the oil and the glass slide. The oil immersion objective magnifies about 100 times.

 

Immersion oil contributes to finer resolution and brightness. These characteristics are most critical under higher magnification. Thus, only the higher power, shorter focus objectives are designed for oil immersion.

 

For any given lens there is a fixed focal length. With the objective in focus there is a cone of light extending from a point on the specimen to the full diameter of the objective lens. Below the specimen is a second, matching cone of light. The base of the cone is the top surface of the condenser and the apex at a point on the specimen. Theoretically, illumination provides a straight line path for each ray from condenser to objective lens. Immersion oil contributes to the homogeneous path of light between the objective and the condenser by having the same index of refraction as the glasses in the system.

 

These objective lens are parfocal. This means that they are designed so that once you have brought an image into focus under low power, you may switch safely to one of the higher powers without hitting the slide or coverslip or making major readjustments with the coarse adjustment. You will find that the object you are viewing will be in approximate focus. If, when switching to a higher power, you meet resistance, check the slide. You may have an unusually thick coverslip or you may have your slide on the stage upside down. Before you move to a higher power, be sure to place the object to be viewed in the center of the present field of view. Remember that as you increase magnification you decrease proportionately the diameter of your field of view.

 

   7. At the upper end of the tube are two oculars or eyepieces. Each contains two lenses. Note

       that they do not fit tightly into the body tube. This is one of the reasons you are asked to carry

       the microscope in an upright position. Note that each ocular bears the marking 10X or 10 on

       the side. This means that it magnifies 10 times. This ocular number, multiplied by the number

       on the objective, gives the approximate magnification obtainable with that objective.

 

It is very important to locate dirt particles on the lens system and to remove them with lens paper or lens paper moistened with alcohol or glass cleaner BEFORE beginning the laboratory work. If a blur appears, a sharp image of the object cannot be obtained until the cause is corrected. As you look through the oculars, turn them. If the smear turns with the ocular, then the ocular is dirty. If it does not turn, try moving the slide. If the dirt is still present, switch the objectives and see if the dirt is on the objective. If not located at any of these points, clean the condenser and/or the built-in light source. In general, clean all surfaces through which light passes.

 

   8. Beneath the stage are a series of lenses which serve to bring the light rays into focus. Known as

       the condenser, it may be raised or lowered by turning the milled knob under the left side of

       the stage (models will vary). To focus the condenser, turn the knob of the light source to a low

       setting. Note that there is only a small spot of light coming from the light source. Put the

       microscope on low power (10X objective) and focus the objective lens on the object on the

       slide. Raise or lower the condenser until a sharp hexagonal shape can be seen through the

       ocular. The condenser is now in focus for this particular slide. In general, moving the condenser

       lens away from the stage causes light to be diffused or spread out. Moving the condenser toward

       the stage brings the light to a point on the slide. As you increase magnification you will normally

       have to move the condenser up towards the stage. Remember, as you increase magnification you

       also must increase illumination.

 

   9. Beneath the stage of the microscope, below the condenser, and sometimes attached to the

       condenser is a series of thin metal leaves which can be opened or closed to regulate the amount

       of light passing through the lens system of the microscope. This is the iris diaphragm.

       The iris diaphragm is opened and closed by moving the lever found under the front-right part of

       the stage. You should adjust this every time that you look at a new object. This will ensure that

       you are achieving maximum contrast.

 

  10. The light source is built into the base of the microscope and controlled by a rheostat knob or

         lever.

 

Proper Use of the Compound Microscope

 

   1. Clean all lens surfaces with lens paper and place the object to be viewed on a slide on the stage

       of the microscope. Be sure that the 10X objective is in position.

 

   2. Adjust the light coming through the microscope so that the object is evenly illuminated and the

       brightness allows for comfort without squinting.

 

   3. Without looking through the microscope, bring the stage up towards the objective until it stops.

       The microscope should have a built-in stop to prevent damage to the lens system at this

       magnification. Move the slide so that the object you are viewing is centered in the circle of

       light coming through the opening in the center of the stage. If no light is present, check to be

       sure the light source is on, the plug is in the electrical outlet, and the diaphragm is open

 

   4. Look through the oculars as you turn the coarse adjustment and lower the stage until the object

       is in focus.

 

   5. Adjust the iris diaphragm and condenser for good lighting.

 

   6. Remember that the objectives on these microscopes are parfocal. Before moving the high 

       power lens into position, move the object to view into the center of the field.

 

Precautions to be Observed when using the Compound Microscope

 

   1. Never touch any of the lens with anything other than lens paper and approved lens cleaner.

 

   2. Always rack down to bring the object into focus.

 

   3. Keep both eyes open at all times.

 

 4. All initial focusing is done with the coarse adjustment. Use the fine adjustment to sharpen

       your final focus and to note the three-dimensional nature of the object in view.

 

   5. Always clean the lenses before and after using the microscope. Be sure to clean all oil from the

       lens after final use.

 

   6. Before returning the microscopes to the cabinet, place the lowest power objective in place. Be

       sure the mechanical stage is not sticking out and be sure that the power cord is not severely

       bent at the point where it leaves the base. Recover the microscopes before replacing them in the

   cabinet.

 

Add picture of binocular microscope Figure 1. Compound Microscope            G045