Background
The microscope is an essential tool that is used to view microscopic structures such as cells and tissues. The microscopes that you will use are expensive, precision instruments that must be handled very carefully. They use visible light and have a magnification range from 40X to
1000X.
When using a microscope, resolution, magnification and contrast are important considerations.
Resolution is the ability to distinguish between two points and therefore, is the ability to see details more clearly. Magnification is an enlargement of the image of the object being examined.
Contrast refers to visible differences in parts of the sample. The wavelength of the light source used in a microscope limits resolution and thus, the magnification that can be achieved. White light used in a microscope limits magnification to 1000 times (1000X) but other microscopes, such as ultra-violet and electron based, can achieve a much higher resolution and magnification.
A simple microscope consists of one lens and provides only one level of image magnification.
Examples include a magnifying glass and a jeweler’s loupe. The stereoscopic (dissecting) microscope is used to view opaque specimens, either alive or preserved. In general, these microscopes can magnify the image 10 or 20 times. Compound microscopes have higher resolution and magnification. Specimens for this type of microscope are usually preserved, thinly sliced or smeared on a slide, and stained to allow light to pass through the specimen and show colored details (resolution and contrast between images). These microscopes magnify the image
40 to 1000 times to allow you to see larger cellular structures. Research labs that investigate subcellular structures such as organelles require a higher magnification (1,000,000X) and resolution. These labs have access to electron microscopes that use an electron beam instead of light (the wavelength of this light is shorter). There are two types of electron microscopes: the transmission electron microscope (TEM), and the scanning electron microscope (SEM). The
TEM uses ultra-thin tissue sections stained with heavy metal ions. The electron beam passes through the specimen and the image is projected onto a fluorescent screen. In the SEM, the electron beam bounces off the surface of the specimen showing enormous surface detail.
Purpose
This laboratory activity introduces working familiarity with the compound microscope, the preparation of simple stained slides and their nuclear observation with other prepared slides.
Materials per team
Compound microscope and Immersion Oil
Prepared slides (Whitefish Blastula, Onion Root Tip)
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Procedures
A. Components of the Bright-Field Compound Microscope
1.
Get a bright-field microscope from the cabinet.
2.
Carry the microscope upright with two hands, one hand supporting the base and the other hand holding the arm.
3.
Go through the following and identify the parts of the microscope (work as a group and work your way from the top of the scope to the bottom – ask me if you have questions).
Oculars (eyepieces). This pair of lenses is used to look into the microscope as well as to magnify the image of the specimen (10X). The distance between the oculars should be adjusted to the appropriate interpupillary distance.
Body tube. The body tube is located between the ocular and the objective lenses, holding the ocular and the revolving nosepiece. It has a mirror inside that reflects the light from the objective lenses to the ocular lenses.
Revolving nosepiece.
It is a revolving metal ring that is located at the bottom of the body tube. It holds the four objective lenses, each of which can be moved into position directly above the opening in the stage. You can feel and/or hear the objectives click into place (try it). If the objective lens is not directly over the opening in the stage, then you will not see a complete field when you look through the microscope.
Objective lenses.
These are the lenses attached to the revolving nosepiece . The objective lenses magnify the image of the object. Bright-field microscopes have four objective lenses and the magnification power is engraved on each lens.

Scanning lens (4x)

Low power lens (10X)

High-dry lens (40X)

Oil immersion lens (100X)
To calculate the total magnification of the image, the power of the objective lens
(variable power) is multiplied by the power of the ocular lens (10X). The scanning and low power lenses are used to initially focus the slide and to give an overview of the specimen. To see greater detail, high-dry and oil-immersion objectives are used.
Stage. This is the platform on which the slide is placed. It is composed of two parts. The stationary stage is where the slide is placed and has an opening to allow light rays to pass through. On top of the stationary stage is the mechanical stage, a movable platform that holds the slide in place. The slide is wedged into the mechanical stage by adjusting the metal calipers. Two knobs below the stage move the mechanical stage and the slide either back and forth or left and right. You will notice that the image moves in the opposite direction from the movement of the slide. This is because the image is inverted and reversed as it passes through the two sets of lenses.
Condenser lens. This unit is located below the stage. It consists of a condenser lens, iris diaphragm, iris diaphragm lever and blue filter. The condenser lens can be seen through the opening of the stage; it focuses light rays coming from the illuminator to the specimen on the stage.
Iris diaphragm and lever. Located under the condenser lens, the iris diaphragm increases or decreases the amount of light coming from the in-base illuminator. It is important to adjust the iris diaphragm when changing slides or when changing magnification since the amount of necessary light will vary the intensity and clarity
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of the image. The position of the condenser lens can be adjusted using the pinion knob.
Pinion knob or condenser lever.
This moves the entire condenser unit up or down to adjust the contrast of the field of view. If the unit is moved closer to the stage, the field of view becomes brighter.
Coarse and fine adjustment knobs. These knobs move either the stage or the body tube.
The image of the object can be focused by increasing or decreasing the distance between the objective and the slide. The larger, outer coarse adjustment knob is used for the initial focusing (4X or 10X objective lenses). The smaller, inner fine adjustment knob is used to precisely focus the specimen (40X or 100X objectives).
In-base illuminator . The microscope lamp is located in the base of the microscope and is usually adjusted by the illuminator rheostat . The intensity of the light should be adjusted depending upon the type of specimen and the intensity of the staining.
Base and arm. The base is the flat, bottom portion of the microscope containing the inbase illuminator, which may have a rheostat. The arm attaches the base to the body tube. When carrying the microscope always use two hands : one hand to support the microscope under its base and the other to hold the arm of the microscope. Be sure to carry the microscope upright.
B . Application of the Bright-Field Compound Microscope
1.
Plug in the microscope (electrical outlet). Do not turn on the light (or turn off the light if it is on).
2.
Clean the lenses with lens paper. Do not use any other type of paper to clean the lens – paper towels will scratch the sensitive lenses .
3.
Turn on the in-base illuminator and adjust the light intensity to 50% with the rheostat.
4.
Turn the revolving nosepiece in a clockwise manner until the 4X objective lens is clicked into place, directly over the opening of the stage.
5.
Grab a prepared slide (Whitefish or Onion) and fit the slide inside the calipers (not
UNDER the calipers). Open the clamp of the slide holder and push the slide all the way into the slot of the slide holder. Release the clamp. Do not put the slide on or under the clamp of the slide holder.
6.
Center the slide by moving the two slide adjustment knobs, located below the stage. The specimen should be in the center of the stage so that the light passes through it.
7.
Turn the coarse adjustment knob slowly until the image comes into view and becomes sharp.
8.
Next, use your hands to move the ocular lenses far apart. Adjust the oculars to the correct interpupillary distance. With both eyes, lock through the ocular lenses. Your eyelashes should not come into contact with the lenses. You should see two bright circular microscopic fields. Adjust the distance between the two ocular lenses until the two microscopic fields overlap to become a single image. This adjustment will be the correct distance between the two ocular lenses; otherwise, you will have double vision
(and a bad headache).
9.
Use the fine adjustment knob to complete focusing the image.
10.
Adjust the incumbent light using one or all of the following methods: adjusting the rheostat, moving the iris diaphragm lever, and raising or lowering the condenser. Usually
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the condenser knob is raised up, and you only need to adjust the other two to get an optimal image.
11.
If a specific part of the image needs to be further magnified, bring that part to the center of the field of view. This step is important because at a higher magnification the field of view covers a smaller part of the slide.
12.
Use the revolving nosepiece to switch to the next higher powered objective. The objective lenses of the microscope are parfocal; the objectives need minimal readjustment when moving from a lower to a higher magnification. Thus, the objective requires only a minimum effort to refocus by using the fine adjustment knob. (You may also need to adjust the intensity of the light by using the rheostat and/or the iris diaphragm).
13.
Since the high power objectives come very close to the slide, it is important to use only
the fine adjustment knob for focusing (otherwise, you can damage the slide and the lens on the microscope).
14.
Although the use of the oil immersion lens is not recommended, follow these steps if told to do so by your instructor. If not, proceed to the next step…

Move the objectives halfway between high dry (40X) and oil immersion (100X)

Add a small drop of oil to the area of the object that needs to be magnified.

Swing the oil immersion lens into position; the lens should come into contact with the oil. Immersion oil is used only for 100X objective lenses, which have a pinhole opening. The oil has a comparable refractive index as the glass. This ensures the continuous pathway of light rays and minimizes light refraction (bending away) so that enough light rays pass through the lens. Immersion oil is not used with other objective lenses as their openings are large enough to allow light rays to pass through without refraction.

Use only the fine adjustment knob for focusing at this magnification . You may have to readjust the contrast and brightness by moving the pinion knob (or the condenser lever) and the iris diaphragm lever simultaneously.
15.
At this point, you would record (draw) the images of the prepared slide at high-dry (40X) or oil-immersion (100X). We will do this soon, but first, complete section A and B of the
Worksheet. After you finish the Worksheet, proceed to the next Exercise, titled,
“Histological Sample Prep.” Bang it out!
16.
When you are done with the microscope, remove the slide. Pre-prepared slides are saved ; student-prepared slides and coverslips are discarded in the Biohazard box.
17.
Clean the oil immersion lens and the slide after you are done by placing a drop of 70% ethanol (or xylene) on a piece of lens paper and gently wiping the lens until there is no more oil residue is left on the lens. Do not use any other type of paper to clean the lens – paper towels will scratch the sensitive lenses . The lens must be cleaned of oil, otherwise the lens will become damaged.
18.
Loosely wrap the electric cord around the base of the microscope before returning it to the cabinet.
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A. Questions
1.
The microscope used to study the internal structures of a cell is a ___________ microscope.
2.
In a compound microscope, the _______________ lens provides the lowest magnification while the ______________ lens gives the highest magnification.
3.
The number engraved on an objective lens represents the ____________ that lens can provide.
4.
What is the magnification of the ocular lens? ____X.
5.
Why do we stain tissue specimens when viewing them under the microscope?
6.
Images observed under the light microscope are reversed and inverted. Explain what this means and why it happens (discuss this with your colleagues). Hint: there is a series of mirrors in a microscope.
7.
If you open the iris diaphragm on low power what happens to the quality of the image? What happens to the image if you close the iris diaphragm?
8.
Complete the following table for the compound microscope:
Objective
Lens
Scanning
Low power
Objective Lens
Power
Ocular Lens
Power
Total
Magnification
High-dry
Oil immersion
9.
Define the term parfocal.
10.
What do you use to clean the oil immersion lens of the compound microscope? Why?
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B. Components of the compound microscope
1.
Using the following list, label the parts of the compound microscope: arm, base, body tube, condenser, course adjustment knob, fine adjustment knob, illuminator rheostat, iris diaphragm, light source, mechanical stage knobs, objective, ocular, pinion knob, revolving nosepiece and stage.
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