Basic Microscopy. OBJECTIVES After completing this exercise, you should be able to do the following:

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1 Page 1 of 10 Basic Microscopy OBJECTIVES After completing this exercise, you should be able to do the following: a. Name the parts of the compound microscope and the functions of each. b. Describe how total magnification is calculated. c. Know the correct procedure for using and caring for the compound microscope. d. Understand the concept of depth of focus. PARTS OF THE MICROSCOPE Read through this information carefully. Be sure you understand each item before proceeding to the next step. 1. Review how to carry a microscope. Obtain a microscope. 2. The major parts of the compound microscope are shown in Figure 1 (at the end of the lab). As you read this text, refer to this diagram and label the parts that appear in bold print. (Your microscope may be slightly different from that illustrated.) 3. The base rests on the table and, in most microscopes, contains a built-in light source. Find the ring around the light source that allows you to open and close the light source diaphragm. Make sure that this diaphragm is open all the way. 4. The arm rises from the base and supports the stage, lens system, and control mechanisms. The stage is the flat surface on which microscope slides are placed for viewing. A mechanical stage holds the slide in place with a clip and can be adjusted side to side and forward and back with stage control knobs. 5. Find the light switch on the arm of the microscope. Turn the light on. Find the dimmer switch and turn it about ½ way between low and high. 6. Your microscope has a condenser located below the stage. It concentrates the light on the object and may be raised or lowered by the condenser control knob. Usually the condenser should be raised to its highest position. Check to see that it is. 7. An iris diaphragm is built into the base of the condenser. The iris diaphragm control lever varies the amount of light entering the condenser and lens system. Move the lever back and forth to see how this works.

2 Page 2 of The body tube is supported by the arm and has a pair of ocular lenses at the upper end. Notice that the ocular lenses move apart and together to adjust for the distance between your eyes. 9. Now find the revolving nosepiece with attached objective lenses at the lower end. The nosepiece is rotated to bring different objectives into viewing position. The objectives will click into place when they are in viewing position. Try this now. 10. The magnification of each objective is indicated on the objective itself. An objective with a magnification of 4x is called a scanning objective. A low-power objective has a magnification of 10x. A high-power objective has a magnification of 40x. Finally, the objective with a magnification of 100x is an oil-immersion objective. 11. Look at your microscope and list the objectives it has here: 12. There are two focusing knobs (one inside the other) that work to move the stage up and down so that it is closer to or farther from the objective lens. The coarsefocusing knob has the largest diameter and is used to bring objects into rough focus when using the 4x and 10x objectives. The fine-focusing knob has a smaller diameter and is used to bring objects into fine focus. It is the ONLY focusing knob used with the high-power and oil-immersion objectives. 13. Check to see that all parts of Figure 1 are labeled. MAGNIFICATION 1. The magnification of a microscope is determined by the power of the ocular and objective lenses being used together. The ocular of your scope has a magnification of 10x. You have a list of the objectives above (in item 11). Total magnification is determined by multiplying the power of the ocular by the power of the objective. 2. Enter the objective and ocular magnifications from your microscope in the table below. Calculate the total magnification when each of the four objectives is being used. OBJECTIVE scanning OBJECTIVE MAGNIFICATION OCULAR MAGNIFICATION TOTAL MAGNIFICATION low-power high-power oil-immersion

3 Page 3 of 10 USING A MICROSCOPE The following exercise is designed to help you develop skill using the compound microscope. Follow the steps and check them off as you go. Write answers to all of the questions. 1. Carry the microscope with two hands: one on the arm and one under the base. 2. Place the microscope on the lab bench with the arm away from you. 3. Use a piece of lens paper to clean the ocular and objectives of your microscope. Only use specially prepared lens paper on the lenses. You could damage the optics using anything other than lens paper. 4. Plug the microscope cord into the outlet at your lab station. 5. Swing the scanning lens into place. You will hear the nosepiece click when the objective is in position. 6. Looking from the side, NOT INTO THE SCOPE, rotate the coarse adjustment knob one-half turn. Rotate it the other way. What happens to the stage? 7. In the same way, rotate the fine adjustment knob one-half turn. Rotate it the other way. How far did the stage move this time compared with the coarse adjustment? 8. Turn the coarse adjustment so that the stage is as near to the scanning objective as possible. Using a piece of paper, mark off the distance between the tip of the objective and the stage. This is a rough estimate of how far the objective will be from the slide. 9. Now swing the low power objective into place, making sure you hear the click. Mark off the distance to the stage using the same piece of paper. Is it larger or smaller than that for the scanning objective?

4 Page 4 of Repeat the measuring procedure after swinging the high power objective into place. Watch to see that it doesn t hit the stage. What happens to the distance between objective and slide as magnification increases? 11. Why should you NEVER USE THE COARSE ADJUSTMENT WHEN USING THE HIGH POWER OBJECTIVE? THE FOLLOWING PROCEDURE IS THE EXACT SEQUENCE OF STEPS THAT YOU ALWAYS FOLLOW WHEN USING A MICROSCOPE! PAY ATTENTION!!! 1. Put the scanning objective into place. 2. Turn on the light of your microscope. 3. Open the light source diaphragm to its widest point. 4. Raise the condenser to its highest position. 5. Looking through the eyepiece, sweep the iris diaphragm control lever all the way from right to left. What happens? 6. Move the iris diaphragm control lever about midway between the extreme left and right positions. You should now have even illumination over the field of vision. The amount of light will have to be adjusted for different specimens and different levels of magnification. 7. Take a slide of the letter e. Place the slide on the mechanical stage so that the clip holds it securely in place. 8. Using the knobs on the mechanical stage, center the letter over the stage opening. 9. Show how the letter e is oriented on the slide as it appears WITHOUT LOOKING THROUGH THE MICROSCOPE by drawing it in Figure 2.

5 Page 5 of 10 Figure 2. Slide of the letter e as seen without using a microscope. 10. Turn the coarse adjustment knob to position the stage as close to the scanning objective as possible. 11. Look through the ocular and adjust the light so that there is even illumination across the whole field of view. 12. Slowly turn the coarse adjustment toward you until you have the letter in view. If after a few turns it doesn t appear, try centering the slide and adjusting the light again. Repeat the focusing procedure. Now use the fine adjustment for getting sharp focus. 13. Compared to the picture that you drew in Figure 2, is the letter e right side up or upside down? 14. Does it face the same way or is it backwards? 15. Using the knobs on the mechanical stage, move the slide to the right slightly. Which way does the image move? 16. Move the slide up. Which way does the image move? 17. In your own words, summarize what happens to the orientation and movement of objects when viewed through the compound microscope.

6 Page 6 of What total magnification are you looking at the letter e at right now? 19. Draw the letter e as you see it in Figure 3. Try to get it about the right size and in the right orientation. Figure 3. Image of the letter e at 40x total magnification 20. Before changing to a different objective, make sure that the object is in the center of your field of view and in sharp focus. Looking at the SIDE OF THE MICROSCOPE, swing the 10x objective into place. 21. Now use the fine adjustment knob to bring the image into sharp focus. You should not have to adjust it more than ½ turn at any time. It may be necessary to increase the light intensity when you use this objective. 22. What total magnification of the letter e are you looking at now? 23. Draw the letter e in Figure 4. Again show the approximate size and orientation. Figure 4. Image of the letter e at 100x total magnification 24. AT THIS POINT HAVE YOUR INSTRUCTOR CHECK YOUR WORK AND INITIAL YOUR PAPER.

7 Page 7 of Looking at the SIDE OF THE MICROSCOPE, carefully swing the high power objective into place. You should not have to touch the adjustment knobs. 26. Remember the coarse adjustment knob is NEVER used with high power. Focus with the fine adjustment knob only. 27. What happened to the light when you changed to high power? 26. Draw what you see in Figure 5. Give a title to this figure. (Use the last 2 figures as examples of how to title your drawing.) Figure Swing the scanning objective back into place then remove the slide. DEPTH OF FOCUS 1. Obtain a thread slide. 2. Using the procedures you learned in the last section, center the crossed threads on the stage. 3. Focus first using the scanning objective, THEN THE LOW POWER OBJECTIVE. 4. Center the threads so that the crossing point is in the middle of your field of view. 5. Move the iris diaphragm control lever so that you have the minimum amount of light coming through but can still see the threads clearly. You should be able to see the smaller fibers that make up each thread. 6. Now position the stage as close to the low power objective as possible using both the coarse and fine adjustments. 7. Looking into the oculars, slowly move the stage down using the coarse and then fine adjustment knobs until the first thread is in sharp focus. What color is the thread?

8 Page 8 of This is the bottom thread on the slide! How do you know this is the bottom? 9. Now continue to move the stage down using the fine adjustment until a second thread appears. What color is it? 10. Does this thread lie over or under the thread you saw in number 6? How do you know this? 11. Now continue to move the stage down using the fine adjustment until a third thread appears. (You may not be able to see all three threads in one place. If this is the case, determine which thread is on bottom and top for each pair of threads. Then, using logic, figure out the order for all three!) What color is it? 12. When one thread is in focus, what is the appearance of the other two threads? 13. The importance of depth of focus lies in the fact that all layers of a specimen on a slide are seldom in focus at the same time. When the upper layer looks sharp, the middle and lower layers are generally blurred. To examine those other layers, we must use the fine adjustment to move the object downward. The vertical distance that remains in focus at any one time is called the depth of focus. 13. CHECK HERE WITH YOUR INSTRUCTOR AND HAVE HER/HIM INITIAL YOUR PAPER.

9 Page 9 of 10 BEFORE PUTTING THE MICROSCOPE AWAY 1. Remove the microscope slide. 2. Turn off the light. 3. Remove the power cord and place it in the designated box. 4. Clean all lenses using lens paper. 5. Move the mechanical stage so that there are no parts sticking out. 6. Rotate the body tube (with the oculars) ½ turn to make the whole microscope as compact as possible. 7. Place the cover on the microscope. 8. Put the microscope in the proper cabinet.

10 Figure 1. Diagram of the microscope Page 10 of 10