Lab Exercise 2: The Microscope

Objectives Once you have completed this exercise, you should be able to do the following: 1. Know the parts of the compound scope and their functions. 2. Use the scope to focus on stained organisms. 3. Use a stage micrometer to measure the field of view (FOV) and estimate size of organisms. 4. Properly clean and store the scope.

Parts of the Compound Microscope Stage This is a platform on which the microscope slide rests and is centered over the light source. The mechanical portion of the stage can be adjusted either vertically or horizontally to center the specimen over the light source. The course and fine focus knobs will raise or lower the stage to focus Light source. A light microscope uses visible light to view the specimen. The intensity of light provided is controlled by both an on/off switch and a light intensity dial. For most experiments in this laboratory, we will have the intensity dial set to maximum. As the magnification increases, the light decreases, so more light is needed. You will have to open the diaphragm or increase the light. Condenser The condenser is composed of 2 sets of lenses found directly below the stage, which collect and concentrate the light upward into the lens systems. The condenser also has an iris diaphragm which can be used to regulate the amount of light entering the lens system. For most experiments in this laboratory, the condenser will be set just a quarter turn short of it’s maximum height (closest to the stage). Use the diaphragm to regulate the amount of light passing through the slide. As a rule of thumb, as the magnification increases, the diaphragm must be opened to allow more light in to view the sample.

Objective Lenses This is the first set of lenses that will magnify the specimen for viewing. These are found on the nosepiece that rotates, which allows you to change the magnification power based the objective in place. For this laboratory, we will use objective lenses which magnify 4x, 10x, 40 x, or 100x. Our microscopes are also what are called parfocal, which means that when one lens is in focus, the others have the same focal length and can be rotated into position without making any major adjustments to achieve focus (usually a half-turn of the fine focus knob will do). The revolving nosepiece is where the objective lenses are attached. This nosepiece allows you to switch objective lenses. Body Tube The body tube attaches to the arm of the microscope and houses the objective lenses and the ocular or eyepiece lenses (see below). On some microscopes the body tube may be raised or lowered by turning the course-adjustment and fine-adjustment knobs. Eyepieces or Ocular Lenses The eyepieces, or ocular lenses, are what are used to view the sample, as well as provide 10x magnification.

Microscopy Principles Total magnification This is achieved by multiplying the ocular lens power by the objective lens power. Thus, the total magnification= 10 x (4 or 10 or 40 or 100). Resolving Power With all microscopes, there is a limit to the extent of magnification that can be achieved. This is dependant on what is called the resolving power of the lens. Resolving power refers to the ability of a lens to show two closely spaced objects as discrete and separate. When magnification increases beyond this point, the image will look blurry. Resolving power is dependant on the wavelength of light used, as well as the numerical aperture of each lens. Smaller wavelengths of light contain more energy and provide better resolving power. Our light microscopes achieve a maximum resolving power (RP) of approximately 0.2 µm using visible light and a maximum objective lens power.

Use and Care of Microscope Below is a basic guideline of steps to begin using your microscope: 1.

Plug in microscope, taking care to insure the cord is firmly attached to the back of the scope. 2. Turn on the light source power switch. SLOWLY increase the light intensity dial until it is at it’s maximum position. 3. Using LENS PAPER ONLY, gently clean the objective lenses. If there is any residual oil remaining from a previous student, please inform instructor.

4. Place microscope slide with specimen on stage. Center the slide over the light source using the stage adjustment knobs. 5. Rotate nosepiece so the 4x or 10x objective lens is in place. Raise stage to the top position. 6. While looking through the ocular lenses, slowly lower the stage using the course adjustment knob until the specimen comes into focus. It is often helpful to very slightly move the stage back and forth during this process. This will help your eye track to the specimen on the side. 7. Use fine focus knob to bring the specimen into sharp focus. 8. Because the microscopes are par focal, you can now move the next objective (i.e. 10x or 40x) into position. Use a half-turn of the fine focus knob only to bring the specimen into sharp focus. Be sure to adjust the iris-diaphragm to the proper setting for the objective lens you are using. 9. Once your specimen is in focus at 40x, you are ready to observe the sample under the oil immersion 100x lens. 10. Rotate the nosepiece so that the slide is between the 40x and 100x lenses. Add one drop of immersion oil directly to the slide. 11. SLOWLY move the 100x lens into place. The 100x lens should be fully immersed in the oil. (NOTE: Once you have added oil to the slide, you can no longer use your 40x objective. If you can not find your specimen, you must rotate to 10x only and not allow the 40x to pass by the slide again, or it will be contaminated with oil.) 12. Using FINE FOCUS ONLY, bring the object into sharp focus.

When done with a microscope you must: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Keep microscope head in normal position Clean any oil off of microscope lens and stage Wrap cord around cord hanger Leave lowest objective in place a. (the red band scanning lens) Lower stage to lowest position Lower the light dimmer to all the way Turn off light Return to proper cabinet Place in cabinet with oculars facing in and handle out

For more information from Nikon on this scope, it’s components and functions go to: http://faculty.sdmiramar.edu/dtrubovitz/microscope/

Lab Exercises Supplies Microscope Prepared slides of eukaryotic microbes Objective micrometer

Protocol 1. Place the objective micrometer on the stage and center in the light path. 2. Focus on the micrometer using the 10x objective. 3. Determine the Field of View (FOV) for the 10x objective - The smallest lines on the stage micrometer are 10 µm apart - Determine how many lines would fit across the field of view (light circle) at 10x 4. You may use the micrometer to measure the FOV for the other objectives as well. - Alternatively you can extrapolate from the FOV for 10x, since the change in magnification is inversely proportional to the change in FOV. - Example: If the magnification increases by 10, the FOV decreases by 10. 5. Remove the micrometer and store carefully in the objective micrometer box. 6. Place prepared slides on the stage, focus, and use your knowledge of FOV to estimate the size of the eukaryotic microbes. 7. Briefly sketch microbes and record any observations. When done with your microscope make sure it is clean and put away properly!

Data and observations 1. Record FOV measurements for each objective lens: Ocular Lens Total Magnification FOV 4x 40x ____________ mm or 10x 100x ____________ mm or 40x 400x ____________ mm or 100x 1000x ____________ mm or

____________ ____________ ____________ ____________

µm µm µm µm

2. Sketch prepared eukaryotic microbes and estimate size based on FOV from above.

Organism name

Name ________________

Estimated size in µm

Drawing

Size _________________

Total magnification ______

Name ________________

Size _________________

Total magnification ______

Name ________________

Size _________________

Total magnification ______

Discussion 1. Describe the position of your hands when carrying the microscope to and from your laboratory bench.

2. For what purpose would you adjust each of the following microscope components during a microscope exercise? a. Iris diaphragm

b. Coarse- adjustment knob

c. Fine-adjustment knob

d. Mechanical stage control

3. Why is it advisable to start first with the low power lens when viewing a slide?

4. Why is it necessary to use oil in conjunction with the 100x lens but not the other objectives?

5. How do light and FOV change when you increase magnification?

6. What is the relationship between FOV and changes in total magnification?