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MBG 113- LAB MANUAL 3
INTRODUCTION TO LIGHT MICROSCOPY
1. INTRODUCTION
Since its invention, the microscope has been a valuable tool in the development of scientific theory. Magnifying lenses have been known for as long as recorded history, but it was not until the advent of the modern compound light microscope that the device was used in biology. A compound microscope is composed of two elements; a primary magnifying lens and a secondary lens system, similar to a telescope. Light is caused to pass through an object and is then focused by the primary and secondary lens. If the beam of light is replaced by an electron beam, the microscope becomes a transmission electron microscope. If light is bounced off of the object instead of passing through, the light microscope becomes a dissecting scope. If electrons are bounced off of the object in a scanned pattern, the instrument becomes a scanning electron microscope. The function of any microscope is to enhance resolution. The microscope is used to create an enlarged view of an object such that we can observe details not otherwise possible with the human eye. Because of the enlargement, resolution is often confused with magnification , which refers to the size of an image. In general, the greater the magnification, the greater the resolution, but this is not always true. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen (Figure 1). At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer. Figure 1. Magnification and resolution are not always the same. On the left, you can see an imaged magnified, but you don’t have better resolution as the image size increase. On the right, you can get better resolution in addition to the magnification.
The reason for the difference between magnification and resolution is the ability of the human eye to see two objects. It is necessary that two objects be about 0.1 mm apart in order for us to detect them as two objects. If they are closer than 0.1 mm, we will perceive them as a single object. If two objects are 0.01 mm apart, we can not detect them unless we magnify an image of them by 10X. What has happened is that we have effectively altered our resolution ability from 0.1 mm to 0.01 mm through the use of a magnifying lens. We would say that our limit of resolution has changed from 0.1 mm to 0.01 mm, or inversely, our resolving power (resolution) has increased by a factor of 10. Unfortunately, a lens can magnify an image without increasing the resolution. Several artifacts can be inherent in the lens design, which causes the objects to become blurry at the edges. Thus, even though they can be made to appear 0.1 mm apart, the edges might be so blurry that we lose the ability to see them as two objects. Think of a standard eye chart: you can see the increased size of a letter, but may be unable to tell what letter is projected. Figure 1 illustrates what can be seen with increased magnification and resolution. If we were to look only at the left side of the figure, we could get the impression that the cell is filled with a homogeneous fluid (cytoplasm). If, however, we look at the right side of the figure, it becomes apparent that the cytoplasm is actually composed of smaller particulate components (chloroplasts, ribosomes, membranes). As we increased the resolution of our microscopes we changed our concepts from protoplasm (the fluid of life) to cytoplasm (the fluid of the cell outside of the nucleus) to a highly ordered machine full of individual organelles. It is readily apparent that while microscope lenses are usually discussed in terms of their magnification, the most important value is their resolution. All microscopes will come with a lens that can magnify 40 times the normal size, but only a quality lens will allow you to see more than you would with a good hand-held magnifying lens. The value for resolution can be measured as the smallest distance between two points, which allows us to see the points as distinct. With this measurement, resolution increases as the distance decreases--that is, there is an inverse correlation between the limit of resolution and what you actually resolve. Limit of resolution = 0. 61 / NA The resolution of a lens is a property of its physical properties and of the wavelength of light that is passed through the lens. The physical properties are summed up in a value known as the numerical aperture (NA) while the wavelength () is determined by the color of light. The numerical aperture of a lens is dependent upon two parameters, the cone angle and the refractive index of the glass of which the lens is composed. The distance of the specimen to the objective lens (working distance) to obtain a proper focus is determined by the objective properties (working distance and focal distance) and the focused light source. The refractive properties of a lens are summed up in a measurement known as the refractive index (n). The refractive index is a function of the bending of light from air through glass and back again. In a microscope, the glass of the lens is specially formulated to increase its refractive index. Once manufactured this property can not be changed. The media around the lens can be altered, however, by removing air from between the objective and the slide, and replacing it with immersion oil. Immersion oil can only be used with those objective lenses specifically designed for the oil. A
2. AIM
To familiarize the students to proper use and care of compound microscopes, to the concept of magnification and resolution, to preparation of wet mount samples, to identification of cells and subcellular structures and specimen staining.
3. PROTOCOL
BEFORE STARTING: RULES WHEN USING A MICROSCOPE
Microscopes are expensive pieces of equipment. Please handle them carefully. There are some basic rules that you need to adhere to when using microscopes. These are:
1. If you have to, always use two hands when moving your microscope. In our microscope you can use both hands to hold the arm. In other microscopes, you might use one hand to hold the arm and the other hand should support the base. 2. Use only LENS PAPER to clean the lenses. Do not use tissues, paper towels, Kimwipes, your shirt, etc. to clean the lenses. Even though these items may feel soft, they can scratch the lenses. 3. The microscope must be on the lowest power objective lens: a. when starting to use the microscope. b. when you finish using the microscope. 4. When you finish using the microscope: a. make sure the objective lens is on the lowest power b. the power is off c. there is no slide on the stage d. if used, the 100x objective is wiped to remove the oil 5. Start focusing your sample at lower magnifications (i.e. 4x, 10x) before increasing the magnification (i.e. 40x, 100X). Note that, these microscopes are parfocal. This means that when the image is in focus on one objective, the image will be in focus with the other objectives. You may only need to fine focus the image for sharpness when the objective lens is changed. Therefore, always, first focus at lower magnifications and then gradually increase the magnification. 6. Never use the course focus knob when using 100x objective lens. Never directly use the 100X. a. Before using 100X, make sure your sample is already in-focus at 40x. b. Then, apply the IMMERSION OIL and switch to 100x. c. Use ONLY the fine focus knob when using 100X objective. d. Wipe the 100x lens clean after usage (ask your assistants how to clean).
3.1. FUNCTIONAL PARTS OF THE COMPOUND MICROSCOPE
Identify the following parts of the microscope on your microscope and label each part on the picture. For your lab manuals, see appendix 1. a. The ocular lens or eyepiece is a 10X lens, which is at the upper end of the tube. The scope that you are using is binocular; it has two eyepieces. b. Nosepiece : the objective lenses are attached below the nosepiece. It allows the user to change the magnification. c. Objective lenses : our scopes have 4 objective lenses attached to a revolving nosepiece. On each lens the magnification/numerical aperture are imprinted. The magnification powers that you will use are: scanning (4X), low (10X), high dry (40X) and oil immersion objective lens (100X). d. Stage and stage clamp : the slide will rest on the stage and will be held in place with the stage clamp. The moveable portion of the stage clamp should only be touching one corner of the slide.
e. Condenser and iris diaphragm lever: The condenser condenses the light rays into a stronger beam. It is located on the front edge beneath the stage. A small lever is behind it to adjust the size of diaphragm. f. Coarse Adjustment Knob: On the left side of the microscope is a large knob use to move the stage up and down to focus the image. This knob is to be only used with the scanning and low power objective lenses. g. Fine Adjustment Knob : Located on both sides of the microscope. Fine adjustment knob is smaller than the coarse adjustment knob. On the left side of the microscope, the fine adjustment knob is located on the coarse adjustment knob. This knob allows for very small changes to the height of the stage. This knob is used to increase the sharp focus of an image and is the only knob to be used with high power. h. Slide Movement Knobs : To the right side of the stage, below the stage, there are two black knobs that you will use to move the slide. One knob will move the slide to the left and right. The other knob will move slide towards and away from you. i. On/Off Switch : Located on right-side of the microscope. j. Light Adjustment Knob : Located below the on/off switch to increase or decrease the light intensity. k. Power cord: Cable with a plug to plug into the power outlet. Supplies electricity for the microscope light source. l. Illuminator: is the light source for the microscope. Our microscopes have a halogen lamp for illumination. m. Microscope arm: The part of the microscope that connects the tube to the base of the microscope.
3.2. MAGNIFICATION POWER AND RESOLUTION LIMIT
The compound microscope utilizes two different sets of lenses to magnify an object. These lenses are the ocular and objective lenses. 1. Determine the magnification for each of the following lenses by looking for the engraved magnification. 2. Calculate the total magnification of object being viewed by multiplying the magnification of the ocular lens and objective lens.
3.4. SIZE AND THE DIAMETER OF THE FIELD OF VIEW
If you know the diameter of the field of view for each magnification, you can estimate the size of the object in that field. 40X and 100X fields may be too small to measure with the ruler, thus you may have to calculate it. 1. Prepare a slide with the “millimeter gridlines” the same way you prepared the letter “e” slide. Make sure the millimeter gridlines is parallel to the side of the slide. 2. Place the slide on the stage and adjust the slide so that the light is transmitted through the gridlines. 3. Start with the 4X lens; and focus the gridlines. 4. Count the number of boxes that can fit across the field of view (or count spaces between the lines). Do not count the lines! Each box is equal to 1 mm. Record this value on the worksheet. Draw what you see through the binocular. 5. Change to 10X lens and count the number of boxes that can fit across the field of view measure the diameter of the field of view. Record this value on the worksheet. Draw what you see through the binocular. 6. Change to 40X lens. What can you see? Can you find the gridlines? Can you estimate the diameter? 7. To determine the field of view under 40X, use this following formula: (Lens 1 diameter) x (Lens 1 magnification) = (Lens 2 diameter) x (Lens 2 magnification) 8. Using the same formula, calculate the field of view under 100X. Do not look at the specimen with 100X! Magnification of the objective lens Number of millimeter boxes seen in the field Measured diameter of the field of view (μm) Calculated diameter of the field of view (μm) 4X Not applicable 10X 40X 100X Not applicable Not applicable
3.5. OBSERVING HUMAN CHEEK EPITHELIAL CELLS
1. Prepare a wet mount of your own cheek epithelial cells as following
4X 10X 40X
( DO NOT TOUCH ANY SLIDE CONTAINING OTHER PEOPLES’ CELLS ):
a. Get a clean slide, cover slip and toothpick. b. Place a drop of water on the slide c. Gently scrape the inside of your cheek and stir your scrapings in to the water drop on the slide. d. Properly dispose the used toothpicks in the designated waste. e. Place a cover slip on the slide at the edge of the “wet” specimen at a 45 angle, and then carefully drop the cover slip onto the specimen minimizing “air bubbles.” f. Compress gently and wipe any excess liquid with tissue paper. Properly dispose the used tissue papers in the designated waste.
2. Having prepared the wet mount, ask your assistants to provide 2-3 drops of methylene blue dye on one side of your slide. 3. Using a torn tissue paper, suck the liquid from the other side of the slide. This will replace the water with the dye. 4. View the stained wet mount of cells under 4X, 10X and 40X in the correct order. Draw the cells and the details carefully at each magnification. What are the structures you see? Why did we stain the cells? What does methylene blue stain? 5. Roughly estimate the average size of the cells, based on your field of view calculations. Compare your estimations with the literature. 6. Carefully remove the slide from the microscope stage and wash it immediately with soap and a lot of tap water. Then, wipe the slides with 70 % ethanol!
4X 10X 40X
45
DO TO AND HINTS FOR THE REPORTS
1. Present the tables, calculations, graphs, and drawings in the results section. You can place the figures in the appendix but you should refer to them in the results section. 2. Make sure you answered all the questions asked in the manual. Most of the questions are asked to lead you in the discussion. Some questions are asked you to be presented in the results section. Try to discriminate experimental results (results section) from experimental interpretations (discussion). a. For example: the questions about the movement direction of the stage and the letter “e” in protocol 3.3 can be summarized in a table and presented in the results section. b. In protocol 3.5, the question of “what are the structures you see?” and “why did we stain the cells? could be written in the results section and also be discussed in the discussion. Whereas, What does methylene blue stain and how and why?” could be discussed in the discussion 3. In the discussion, compare your results with expected results, whenever possible. (i.e. estimated sizes of human epithelium, bacteria and yeast. ) 4. In the introduction, write about different types of microscopes and their usages, advantages and disadvantages. 5. In the discussion, discuss about the resolution and magnification, based on your results. Compare the size of the cells you observed and give a little background about the cell types. 6. Discuss what you saw and what you could not see.
APPENDIX 1:
Please label the parts described in Protocol 1 and attach this paper to your report as APPENDIX 1
