Lab 5 Microscopy Introduction The microscope is an instrument that contains one or more lenses and is used to view objects that are too small be seen with the unaided eye. A magnifying glass is a simple microscope because it contains only one lens. A lens functions by refracting (bending) light rays coming from an object and focusing them to from an image of the object. A compound microscope contains more than one lens. The microscope is useful for making observations and collecting data in scientific experiments. Microscopy involves three basic concepts: 1. Magnification - The amount that the image of the specimen is enlarged. 2. Resolving power - How well detail is observed and preserved during the magnification process. 3. Contrast - The ability to observe detail against the background. Often, stains and dyes are used on biological specimens to increase contrast. Sometimes special optics are used such as phase contrast lenses. Parts of the Compound Microscope The bottom of the microscope is the called the base. Mounted on the base is the fixed light source. Light then passes through the diaphragm (iris, aperture) which you can use to adjust the amount of light coming from the light source. After the diaphragm, the light then passes through the condenser which focuses the light on the specimen. The object (specimen) to be examined is placed on the stage. Light passing through the specimen then enters the objective lens. The objective lenses are attached to the revolving nosepiece which facilitates changing from one objective lens power to another by revolving upon its axis. Our microscopes have three objective lenses: the scanning or low power lens (10X), high power (40X), and oil immersion (100X). We will not use the oil immersion lens in this class. After the objective lens, light then passes up the body tube. At the upper end of the body tube is the ocular lens which focuses the light on the retina of your eye. The magnification power of the ocular lens is 10X. Focusing the microscope: There are two round knobs on the arm of the microscope. The larger knob is the coarse adjustment knob. This knob is used to make large, coarse adjustments when focusing on a specimen. Use the coarse adjustment knob when using the low power objective lens only. The lower smaller knob is the fine adjustment knob. Manipulating this knob makes fine adjustments when focusing on a specimen. Carrying the Microscope The microscope is an expensive instrument. When removing the microscope from the storage cabinet, always grasp it with both hands. Place one hand around the arm and the other hand firmly under the base. Hold it close to your body for stability. Once you reach the lab bench, set the microscope down gently on the table. Do not slide the microscope across the table. Instead, lift it off the table and gently place it back down where you want it. Depth of Focus The vertical distance that stays in focus at a single time is called the focal plane. On high power, the focal plane is more shallow than on low power. As you focus up and down, you will get an idea of the specimen s threedimensional form. Magnification The magnification of each objective lens is marked on the side of the lens. These lenses are color coded. The scanning lens (10X) has a yellow ring on it, the high power (40X) lens has a blue ring, and the oil immersion lens (100X) has a black ring on it. The ocular lens has a magnification of 10X. The total magnification is obtained by multiplying the power of the ocular eye piece (10X) by the power of the objective lens used. Lab 5 Page 1
Using the Microscope First make sure the scanning (low power) lens is in position. (NOTE: this is the way the microscope is stored.) Next place the prepared slide on the stage in such a position that the object of interest is centered over the hole in the stage. With the scanning lens (low power), there is little danger of smashing a slide; nevertheless, make sure the objective lens does not touch the slide. Turn on the light. Look into the ocular lens. The field should be evenly illuminated and bright. You are now ready to focus the microscope. While looking through the ocular lens, focus slowly by turning the course adjustment until the object is brought into view. If you continue to focus and the object does not come into view, you have passed the proper focal plane and must start over again. If after two or three attempts you are still unable to focus on the slide, call your instructor for help. The focus may be sharpened with the fine adjustment if necessary. When changing from a lower power to a higher power objective, very little adjustment is necessary. This is due to the fact that these microscopes are parfocal, meaning that the subject remains nearly in focus from one objective lens to the next. Therefore, only use the coarse adjustment with the scanning or low power lens. Never use the coarse adjustment when using the high power or oil immersion lens. Not only is it unnecessary due to the parfocality of the instrument, but you also risk cracking a slide or an expensive objective lens. The distance between the objective lens and the slide decreases with increased magnification. Change to the high power objective by revolving the nosepiece until the object is in line with the tube. CAUTION: Keep your head to the side to be sure that the lens does not smash the slide! The lens is properly seated when you hear and/or feel the click of the indent stop. You may have to twist the fine adjustment knob to sharpen the image by turning slightly upward or downward. Now that you know what to do, practice will enable you to use the microscope with ease. Remember, start with low power and scan for objects on the slide. Do not hunt with high power. To avoid eye strain, practice using the microscope with both eyes open. This is most easily accomplished by covering the eye not in use with your hand, and then removing your hand while leaving your eye in neutral. With practice you will soon learn to leave your eye in neutral when using the microscope. Make sure the microscope is clean at all times. Keep your fingers off the ocular and objective lenses. If there is dirt on any of the lenses, it must be removed with lens tissue paper only! Exercise 5.1 Specimen Orientation 1. Obtain a permanent slide with the letter e on it. Carefully looking at the slide with your unaided eye determine if e is right side up or upside down. Remembering the orientation of the letter, center the e in the field if view and carefully bring it into focus under the scanning lens. 2. Draw the e as you observe it through the microscope below. 3. Rotate the nosepiece to the high power (40X) objective lens and focus with the fine focus only. 4. Again draw the e as you observe it now. 5. With either objective lens in place, move the prepared slide to the right while watching the image through the microscope. 6. Repeat step 5 only move the slide away from you. Lab 5 Page 2
Exercise 5.1 Report Name 1. Calculate the total magnification for the combined ocular lens and objective lens in the table below. Objective Lens Lens Power Ring Color on lens Ocular lens power Unaided eye 1X none 1X Total Magnification Scanning 4X red 10X 4X 10X = 40X Low Power 10X yellow 10X High Power 40X blue 10X Oil Immersion 100X black 10X 2. Draw the e as seen under low and high power. Calculate the total magnification of each field and write it on the line provided. 3. When you moved the slide to the right which way did the image in the microscope move? 4. When you moved the slide away from you which way did the image in the microscope move? 5. What is the relationship between the movement of the image and the movement of the specimen? Lab 5 Page 3
Exercise 5.2 Report 1. What is the order of threads on the prepared slide? Name Top Middle Bottom 2. How did you determine the order of threads? 3. What diaphragm adjustment can be made to more easily determine the order of the colored threads? Exercise 5.3 Report 1. Draw & measure the diameter of your field of view using a Stage Micrometer. Low Power High Power 2. What is the diameter of the field of view under low power? 3. What is the diameter of the field of view you can observe under high power? Exercise 5.4 Report 1. Draw the image of a cheek cell under high power in the field diagram to the right. Lab 5 Page 4
Returning the Microscope. When you are finished using the microscope, it must be returned to the storage cabinet in the proper condition. It should be clean. If the lenses are dirty, obtain lens paper from the cabinet and clean them. Place the lowest power objective (yellow ring) in the light path. Move the body tube down as far as it will go (or it will not fit into the cabinet). Turn the light off. Wrap the electrical cord loosely around the base. Cover the microscope with the dust cover. Place the microscope (carrying it with two hands) back in the correctly numbered compartment of the storage cabinet. Exercise 5.2 Depth of Focus 1. Obtain a slide with three colored threads mounted one over another. 2. Using low power (10X), find the point where all three threads cross. Then switch to high power (40X). 3. Slowly focus up and down with the fine adjustment knob to observe the depth of the threads. 4. Notice that when one thread is in focus the others seem blurred. 5. Determine the order of the colored threads and enter the results below. Exercise 5.3 Field of View 1. Obtain a microscope slide with a ruled stage-micrometer. 2. Draw what you observe under both low and high power and determine the diameter of each field. Exercise 5.4 Viewing Cheek Cells 1. Obtain a new microscope slide. 2. Place a drop of Methylene Blue stain in the middle of the slide. 3. Use a sterile toothpick and gently scrape the inside of your cheek with the flattened end of the toothpick. 4. Swirl the flat end of the toothpick in the drop of stain on the microscope slide. 5. Obtain a cover slip and place it over the drop on the slide. 6. Observe the cells under the microscope under low power then switch to high power. 7. Under high power find the nucleus, a round centrally located body within each cell. 8. Draw the high power image on your lab report. Lab 5 Page 5
The Microscope Label the parts of the microscope below. Lab 5 Page 6