AN INTRODUCTION TO THE MICROSCOPE

AN INTRODUCTION TO THE MICROSCOPE INTRODUCTION In this exercise you will learn the components and operation of the compound microscope and the dissection microscope. This will be followed by a short exercise in which you will use the microscope to examine plant and animal cells that are actively dividing. OBJECTIVES: Learn the parts of the compound microscope. Learn the operation of the compound microscope. Learn and apply the following microscope concepts: Total Magnification, Depth of Field, and Field of View. Practice with the compound microscope. PART 1: THE PARTS AND PIECES OF THE COMPOUND MICROSCOPE You will begin by securing a compound microscope. Always carry a microscope with two hands. Your instructor will demonstrate the accepted way of carrying a microscope and the particular rules governing their use in your laboratory classroom. 25Jan17 2:23 pm Microscope - 1

The Parts & Pieces (continued) Use the generalized compound microscope diagram on the preceding page along with the following descriptions to learn the parts and functions of the compound microscope. 1. The flat area upon which the specimen is placed is called the STAGE. 2. The EYEPIECES or OCULARS are used to view the specimen. Is your scope Binocular or Monocular? 3. The NECK, ARM or SPINE supports the ocular head 4. The LIGHT SOURCE is used to provide illumination. Since the Light Source comes from the bottom through the specimen to the lens, all specimens viewed through a compound scope must be translucent. The amount of light can be controlled by adjusting the intensity knob mounted in the base. 5. The IRIS DIAPHRAGM is mounted in the CONDENSER (not shown in diagram) which is located just below the stage. The Iris diaphragm is used to adjust the amount of light passing from the light source through the specimen. The height of the condenser is controlled by a small knob below the stage. IF NOT ALREADY ADJUSTED TO THE HIGHEST SETTING, ROTATE THE CONDENSER KNOB TO RAISE THE CONDENSER SO IT IS JUST AGAINST THE LOWER SIDE OF THE STAGE. The Iris Diaphragm has a control rod that is used to open or close the opening. Familiarize yourself with the control rod and its the fully open and fully closed settings. Begin each microscope session by ensuring that the Iris Diaphragm is on the closed setting. Less light means more detail so always use the least amount of light possible to view specimens 6. There are two nested FOCUS ADJUSTMENT KNOBS. The outer, larger knob is the COURSE ADJUSTMENT FOCUS knob. The smaller, inner knob is the FINE ADJUSTMENT FOCUS knob. 25Jan17 2:23 pm Microscope - 2

PART 2: USING THE COMPOUND MICROSCOPE #1 - Find the OCULAR magnification for your compound scope and write it in the space provided:. The scopes you will be using are BINOCULAR. Not all compound scopes are binocular. Unlike dissection scopes, binocular eyepieces on the compound scope do not result in threedimensional viewing. Their purpose is to make the scope easier to use. Binocular eyepieces are used because people commonly look at things with both eyes. Binocular scopes, in general, are not better or worse from a performance standpoint than Monocular scopes. #2 - Find the OBJECTIVES and make note of the number and magnification. Your scope should have 3 objectives. # of objectives: magnifications: 2a. Each objective is given a specific name based on its magnification. Match each magnification above to a lens name by writing in the magnification number in the space provided. Description of Lens The lowest power objective The next higher power The next higher power Magnification Number Lens Name SCANNING LENS LOW POWER LENS HIGH-DRY LENS 2b. Determining TOTAL MAGNIFICATION - The Total Magnification of the specimen being viewed is the PRODUCT OF THE OCULAR LENS MAGNIFICATION AND THE OBJECTIVE LENS MAGNIFICATION. Total Magnification = Ocular Lens X Objective Lens Determine the Total Magnification for each objective lens. Lens Total Magnification Scanning Lens Low Power Lens High-Dry Lens 25Jan17 2:23 pm Microscope - 3

Using the Compound Scope (continued) #3 - Secure a letter "e" slide from the front of the room. Adjust the objectives so that the Scanning Lens is locked into the viewing position. YOU SHOULD ALWAYS BEGIN ANY MICROSCOPE EXAMINATION WITH THE SCANNING LENS. Place the slide on the stage and secure it with the slide holder. The slide should sit flat on the stage and be held in place along its two short sides and the lower long side by the slide holder. The scopes you will use are equipped with mechanical stages. These stages allow you to move the slide up, down, left, and right with relative precision. The knobs controlling the movements are either mounted above the stage or below the stage. Find the control knobs on your mechanical stage. Experiment with the knobs to discover which controls up/down movement and which controls left/right movement. DO NOT LOOK THROUGH THE SCOPE AS YOU ARE DOING THIS. Once you are familiar with the controls, proceed to the next section. 3a. Position the slide so that the letter "e" is in its correct orientation on the stage. View the letter through the scope. Adjust the COARSE ADJUSTMENT KNOB to bring the letter into focus. If you are having trouble seeing the object, adjust the light entering the specimen by using the IRIS DIAPHRAGM. ALLOWING TOO MUCH LIGHT THROUGH THE DIAPHRAGM IS THE MISTAKE MOST OFTEN MADE BY INEXPERIENCED MICROSCOPE USERS. This causes the specimen to be either unviewable or eliminates most of the detail. IT IS BEST TO START AT SCANNING POWER WITH THE LEAST AMOUNT OF LIGHT ENTERING THE SPECIMEN. You can then gradually open the diaphragm as you increase the magnification power. Use the mechanical stage knobs to position the letter in the center of the field of view. Did the orientation of the letter change when viewing it through the scope? If so, how? 25Jan17 2:23 pm Microscope - 4

Using the Compound Scope (continued) #3a. (con't) Draw a diagram of the letter on the stage and through the scope. For the scope view, make the size and field of view position as accurate as possible. On the Stage: Through the Scope: 3b. Move the specimen to the right and left and up and down while observing it through the scope. Does the specimen move the same way on the stage and when viewing it through the scope? If not, what differences do you notice? 3c. While viewing the specimen through the scope, increase the magnification by rotating and locking new objectives into place. Stop at the High-Dry Lens. What happens to the amount of the specimen you can view as the magnification increases? 3d. The viewing area is called the FIELD OF VIEW (FOV). The FOV determines how much of the specimen is viewable. What is the relationship between FOV and magnification? (direct or inverse) 25Jan17 2:23 pm Microscope - 5

Using the Compound Scope (continued) #3d. (con't) Rotate the objective back to the Scanning Lens. Remove the slide and return it to the table. Secure a translucent 15cm ruler. Place the ruler on the stage. Each of the graduations on the ruler is 1mm. How many millimeters can you see across the field of view at scanning power? low power? high power? (Note: As the power increases the bars between the graduations will get wider. Take this into consideration when making estimates) Diagram the view at each power. Based on your observations: What is the Field of View at Scanning Power (in mm)? What is the Field of View at Low Power (in mm)? What is the Field of View at High Power (in mm)? As you have observed, it is difficult to estimate the FOV at high power since it is less than 1mm. However, it is possible to calculate the FOV at high power by using the information you have about the FOV at low power. Since each microscope is different, a sample calculation has been done below. Adjust the numbers for your scope's objective lens and low power FOV. Field of View at Low Power = FOV LOW = 1.1mm Field of View at High Power = FOV HIGH =?????? Low Power Objective = OBJ LOW = 10x High Power Objective = OBJ HIGH = 40x Formula: Sample Calculation: 25Jan17 2:23 pm Microscope - 6

Using the Compound Scope (continued) #3d. (con't) Now, do the same calculation for your microscope. Enter your estimated measurement of the FOV LOW in the space below AND use this information and the formula on page 6 to calculate your FOV HIGH Calculation: FOV LOW = Field of View measurements are used to estimate the size of objects being viewed with the compound scope. Keep the Field of View measurements in mind as you use the scope during the rest of this lab exercise and others that follow. #3e. As you just calculated, FOV HIGH measurements are less than one (1) mm. As objects get smaller, scientists will often switch to successively smaller units of measure. The most common unit of measure for cells and subcellular structures is the micron symbolized by the greek letter mu (ì). One (1) micron = 10-3 mm = 0.001mm One (1) mm = 1000 ì Refer back to part #3d and convert the example FOV HIGH (at the bottom of pg.6) and your calculated FOV HIGH from mm to microns. 0.4375 mm = ì Calculated FOV HIGH = mm = ì Return the ruler to the front table. 25Jan17 2:23 pm Microscope - 7

PART 3: PRACTICE WITH THE COMPOUND MICROSCOPE In this part you will make some temporary microscope slides and view them using the skills learned in Part 2. Making and Viewing Wet Mounts 1. Secure a clean, dry microscope slide, a coverslip, and a toothpick. 2. Rub the toothpick along the inner surface of your cheek. You will notice a white, filmy substance coating the toothpick after the process. This material contains living cheek cells. 3. Scrape the toothpick along the center of the microscope slide to deposit some of the cells on the slide. Add 1 drop of stain and 1 drop of water then place the coverslip on top. 4. Set the scanning lens in place and insert the slide onto the microscope. 5. Examine the cells under scanning, low, and high power. Make a sketch of the field of view at each magnification. 6. Using your FOV measurements and calculations to estimate the size of a cheek cell in microns. 7. Clean the slide and coverslip in preparation for the next wet mount. 25Jan17 2:23 pm Microscope - 8

Part 3: Practice with the Compound Microscope (con t) Making Wet Mounts (con t) 8. Secure a single Elodea leaf. Elodea is small water plant often used in fresh-water aquaria. 9. Place the leaf on the slide with a few drops of water and a coverslip. a. Focus using the Coarse Adjustment. b. Using the Fine Adjustment, focus up and down repeatedly. Notice how different layers of the specimen come into view. This phenomenon is called DEPTH OF FIELD. When you view a specimen with a compound microscope, only a "slice" of that specimen can be in focus at any one time. The lower the magnification, the deeper the Depth of Field. In other words, at low powers you view a "thicker" slice of the specimen than at higher powers. c. View the specimen at progressively higher powers. At each power, notice the difference in the amount of Fine Adjustment movement that is necessary to move between layers. The Depth of Field is becoming shallower as you increase the magnification. If you notice a reduction in clarity as you increase the magnification, adjust the iris diaphragm. 10. Make a sketch of the field of view at each magnification. 11. Using your FOV measurements and calculations to estimate the size of the long and short axis of an Elodea leaf cell in microns:. 25Jan17 2:23 pm Microscope - 9