AP BIOLOGY Cells ACTIVITY #2 MICROSCOPE LAB OBJECTIVES 1. Demonstrate proper care and use of a compound microscope. 2. Identify the parts of the microscope and describe the function of each part. 3. Compare magnification, resolving power, and contrast. 4. Demonstrate proper technique of preparing a wet mount slide. 5. Demonstrate inversion and depth of field. 6. Use the compound microscope as an instrument of measurement. INTRODUCTION The unaided human eye can detect objects as small as 0.1 mm in diameter. Most cells are between 0.01 mm and 0.1 mm in diameter and cannot be seen without a microscope. A microscope contains one or more lenses and is used to view detail that cannot be seen with the unaided eye. The light microscope, by virtue of its lens system, extends our vision a thousand times so that object as small as 0.1 micrometer (μm) in diameter can be seen. The electron microscope further extends our viewing capability down to 1 nanometer (nm). At this magnification it is possible to see a virus and the outline of individual protein or nucleic acid molecules. A lens functions by refracting (bending) light rays coming from an object and focusing them to form an image of that object. Refraction of light is due to the angle at which it passes from one transparent medium to another (for example, air to glass) and the difference in density between the media. A magnifying glass is a simple light microscope. The microscope consists of a set of lenses that focus an enlarged image of an object on the retina of the eye. BASIC MICROSCOPY The microscope is useful in making observations and collecting data in scientific experiments. Microscopy involves three basic concepts: Magnification The degree to which the image of a specimen is enlarged. Resolving Power How well specimen detail is preserved during the magnifying process. Contrast The ability to see specimen detail against its background. Stains and dyes are added to sections of biological specimens to increase contrast. Cell Activity #2 Page 1 of 10
BASIC MICROSCOPE LAYOUT *Always keep the eyepiece facing away from the direction the stage faces. *Never move the microscope to show your partner something. Instead- switch him or her places. *Never put away a microscope that is dirty or has a slide on the stage. Cell Activity #2 Page 2 of 10
Abbe Condenser: A specially designed lens that mounts under the stage and is usually movable in the vertical direction. It has an iris type aperture to control the diameter of the beam of light entering the lens system. By changing the size of the iris and moving the lens toward or away from the stage, the diameter and focal point of the cone of light that goes through the specimen can be controlled. Abbe condensers really become useful at magnifications above 400X. Arm: The part of the microscope that connects the tube to the base. When carrying a microscope, grab the arm with one hand and place your other hand under the base. Base: The bottom support of the microscope (see arm above). Coarse Focus: This is the rough focus knob on the microscope. You use it to move the objective lenses toward or away from the specimen (see fine focus). Do not use this if you are in HIGH POWER. You could crack the slide. Condenser Lens: A lens mounted in or below the stage whose purpose is to focus or condense the light onto the specimen. By using a condenser lens you will increase the Illumination and resolution. Condenser lenses are not required on low power microscopes. Cover Slip: A very thin square piece of glass or plastic placed over the specimen on a microscope slide. When used with liquid samples, it flattens out the liquid and assists with single plane focusing. Usually these are disposable. Diaphragm: Not present on these scopes. Eyepiece Lens: The lens at the top of the microscope that you look into. They are usually 10X but also are available in 5X, 15X and 20X. Fine Focus: This is the knob used to fine tune the focus on the specimen. It is also used to focus on various parts of the specimen. Generally one uses the coarse focus first to get close then move to the fine focus knob for fine tuning. If you turn this many many times you will really mess it up. It should not take many turns to find focus. Field of View: Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see when looking into a microscope. As the power gets greater, the field of view gets smaller. You can measure this by placing a clear metric ruler on the stage and counting the millimeters from one side to the other. Head: The upper part of the microscope that contains the eyepiece tube and prisms. A monocular head has one eyepiece, a binocular has two (one for each eye). We have monocular heads. Illuminator: A light source mounted under the stage. Our light source has a variable output instead of having a diaphragm under the stage and a fixed lamp. Mechanical Stage: A mechanical way to move the slide around on your stage. It consists of a slide holder and two knobs. Turn one knob and the slide moves toward or away from you. Turn the other knob and the slide moves left and right. Since everything is upside down on a (high power) microscope it takes some getting used to but it is very convenient to have one especially when observing moving specimens like protozoans or other pond water critters. Place the slide in the Mechanical Specimen Holder. Nosepiece: The part of the microscope that holds the objective lenses also called a revolving nosepiece or turret. Objective Lens: The lens closest to the object. We have three (4X, 10X, and 40X) Parfocal: This is a focus issue. When changing from one objective to another, the new image should be either in focus or close enough so that you can refocus with only minor adjustments. All of our microscopes are parfocaled. Pointer: When you look through the eyepiece lens, you may see a pointer. By turning the eyepiece, you can rotate the pointer around. Slide: A flat glass or plastic rectangular plate that the specimen is placed on. It may have a depression or well to hold a few drops of liquid. Stage: The flat plate where the slides are placed for observation. X: Times as in 200X or two hundred times magnification. The magnification of a microscope is determined by multiplying the power of the eyepiece lens by the power of the corresponding objective lens Cell Activity #2 Page 3 of 10
Size of Objective Magnification Eyepiece Mag. Total Magnification Smallest (Scanning) Medium Largest (High) Not Present (Oil Immersion) 100X 10X 10X 10X 10X Part A. General Microscopy Procedure 1. Obtain a prepared slide from the supply area. 2. Make sure the scanning power lens is in place. (4X) and turn on the power to the light source. 3. Turn up the light source intensity to about 75% or full range of dial. 4. Open up the iris fully to the left (toward the X&Y adjustment knobs) 5. Lower the stage with the course adjustment knob so you can place the slide on the stage without hitting the objective lens. 6. Place the slide on the stage of your microscope and clip it into place. Turn the mechanical X & Y stage position knobs until the center of the slide is right over the light source (circular lens below the stage). 7. While looking at the microscope from the side, move the stage all the way up using the course adjustment knob. 8. While looking through the eyepiece, turn the course adjustment knob away from you until the specimen is in focus 9. Adjust the light intensity and iris until you have the best view of the specimen. 10. With the specimen in focus and positioned in the center of the field of view, rotate the nosepiece lens to the medium power objective (10X). DO NOT move the coarse focus. Only fine focus should be necessary to bring the specimen into sharp focus. Remember: The ability of the microscope t remain in focus when switching from one objective lens to the next highest power is called parfocal. 11. With the specimen in focus and positioned in the center of the field of view, rotate the nosepiece lens to the high power objective (40X). DO NOT move the coarse focus. Only fine focus should be necessary to bring the specimen into sharp focus. 12. Draw a pencil sketch of what you see in the front of your lab book. Be sure to note the name of the slide, total magnification, topic (in this case microcopy), and date. Cell Activity #2 Page 4 of 10
13. Add color with colored pencils or crayons to make it look realistic. Consult your textbook, lab manual, and/or - at last resort your teacher to see if you have the right details. 14. Label all visible structures. 15. Return microscope to scanning power, remove the slide and return it to the tray, and let your partner do the next one! Both partners draw both specimens. 16. This sequence of events should be repeated every time you get a new specimen slide! Part B. Specimen Orientation Procedure 1. Cut out a typed lowercase letter e from a scrap sheet of paper. 2. Place the e in the center of a microscope slide 3. Place the e slide on the stage so that the bottom of the e is facing the arm of the microscope. 4. Make sure the scanning power lens is in place. (4X) and use the procedure (above) to find the e in scanning power. 5. Sketch what you see in your lab notebook. Label it e and 40X What do you notice about the e? (What does the lens do to the image?) When looking through the eyepiece, what happens when you move the slide forward? When looking through the eyepiece, what happens when you move the slide back? When looking through the eyepiece, what happens when you move the slide left? When looking through the eyepiece, what happens when you move the slide right? 6. Repeat at medium power (including sketch). 7. Repeat at High power (including sketch). You may only be able to sketch PART of the image. Use more detail under HIGH. Part C. Field of View Measurement Procedure Most of the objects you view under the compound microscope are smaller than two millimeters. Obviously, measuring these microscopic objects could prove to be quite difficult and inexact if millimeters are used as the unit of measure. To solve this problem scientists divide the millimeter into 1000 smaller units called micrometers (μm). Tiny objects can then be accurately measured in micrometers. In this section you will learn how to estimate the size of the tiny organisms you view under the compound microscope. Obtain a transparent plastic ruler from the supply area. 1. Place the plastic ruler on the stage so that the ruler s edge is centered in your field of view under low power. Make sure you use the millimeter side of the ruler. Use the diagram for help. Cell Activity #2 Page 5 of 10
2. Look through the eyepiece Position the ruler so one of the millimeter marks is just visible to the left in your field of view. Use the diagram for help. Notice the distance between the mark on the left and the next mark is one millimeter. Estimate the remaining distance in decimal fractions of a millimeter across the diameter of the field of view. What is your total field of view size in millimeters under scanning power? mm 3. What is the scanning power field diameter in micrometers (μm)? (1 mm = 1000 μm) μm 4. What is your total field of view size in millimeters under medium power? mm 5. What is the medium power field diameter in micrometers (μm)? (1 mm = 1000 μm) μm 6. Switch to high power. Look at the marks on the ruler. You will find that the high power field of view is less than 1mm or 1000μm. For that reason, it is difficult to estimate the diameter of the field of view using the same technique used for low power. However, you can determine the field of view under high power by using the formula below: 7. Using the simple algebra formula above What is the high power field diameter in micrometers (μm)? (1 mm = 1000 μm) μm 8. Now that you know the diameter of your field size under both high and low power, you can use that information to estimate the size of objects you observe under the microscope. For example, in the diagram at the right, 10 circular objects fit across the field of view. The field of view is 2000μm in diameter. Since each object takes up 1/10 of the 2000μm field diameter, the size of each object is 200μm. You can use this method to estimate size of objects you view under your microscope once you know your microscope s field diameter. Cell Activity #2 Page 6 of 10
9. Obtain three prepared slides from the tray and use the methods (above) to estimate the size or thickness of the objects. Record your findings in the data table. Magnification? Number of Estimated Field Specimen Name Whichever allows you to est. Specimens that fit specimen size or size best. Diameter across thickness Part D. Depth of Field Procedure 1. Obtain a slide of colored threads from the supply area. 2. Look at the slide under low power where the threads cross. Adjust the knobs to give the sharpest view. Are all three thread colors equally visible under low power? 3. Look at the slide under high power where the threads cross. Adjust the knobs to give the sharpest view. Are all three thread colors equally visible under low power? 4. Slowly fine focus up and down to determine the order of the thread colors. a) Top b) Middle c) Bottom *no drawings necessary The Wiggle Technique This will come in handy in the future! 1. Sometimes a slide will be a very thin section of a tissue. In this case you must not let the light wash out the specimen. Put the light very low and close the iris. 2. Once you place the specimen right in the middle, wiggle the slide back and forth slightly while focusing with the course adjustment knob. When you get close you will start to see the slide moving back and forth in your field of view. If you see no back and forth then you are not close to being in focus! *no drawings necessary Cell Activity #2 Page 7 of 10
QUESTIONS 1. Identify the parts of the microscope from memory. a. b. c. d. e. f. g. h. i. j. k. l. m. 2. Describe how a compound microscope should be held and carried. 3. How is the total magnification of a microscope determined? 4. If the eyepiece on a microscope has a magnification of 10X, what is the total magnification with a 15X objective? 5. If the eyepiece on a microscope has a magnification of 15X, what is the total magnification with a 45X objective? 6. An image is located in the lower right hand corner of the field of view. How would you move the slide to center the image? 7. Objects viewed under a compound microscope are frequently lost when switching from low to high power. Give one reason why this happens. Cell Activity #2 Page 8 of 10
8. How did the light intensity change when you switched from low power to high power objective? 9. Do you observe more or less area in your field of view when under high power compared to low power? 10. If a microscope has a low power magnification of 100X and a high power magnification of 500X, and a low power field of 1500μm, what is the high power field in μm? (SHOW YOUR WORK) 11. If 20 objects fit across the diameter of a low power field of view whose field diameter is 4000μm, what would be the approximate size of each object? 12. Why is it more difficult to measure the diameter of the high power field of view than the low power field of view? 13. The circle at the right represents a microscope s field of view with a black dot under 10X magnification. Draw how large the dot would appear under 40X magnification. Also, draw a circle to indicate the size of the field of view under 40X 14. Sketch the number 4 as it appears through the lenses of the compound microscope. 15. How has the lens system of the compound microscope changed the orientation of the numeral? 16. A student focuses on a specimen at low power and carefully centers it before changing to high power. At high power, however, he doesn t see the part of the specimen he was interested in. What might be the problem? 17. Inspired by her biology lab, a student decides to make a closer study of the food she eats. She uses a razor blade to make a very thin section from a raw potato and mounts it in a drop of water on a slide. To her disappointment, she can barely make out the cells under the microscope. What might she do to improve her results? Cell Activity #2 Page 9 of 10
18. How is magnification different from resolving power? 19. What are the advantages and limitations of studying cells using light microscopy? 20. Compare and contrast transmission electron microscopy (TEM) and scanning electron microscopy (SEM). 21. What are the advantages and limitations of studying cells using electron microscopy? Cell Activity #2 Page 10 of 10