Introduction to Microscopes

Size: px

Start display at page:

Download "Introduction to Microscopes"

David Rice
6 years ago
Views:

1 INTRODUCTION TO THE MICROSCOPE Introduction to Microscopes The first microscopes worked by the same basic principle as the ones you will be using in lab. They are light microscopes. Visible light passes through the specimen and then through glass lenses. The light is refracted (bent) by the lenses in order to magnify the image and then projected into the eye. There are two important qualities of a microscope: magnification and resolving power. Magnification is how large an image appears in comparison to its actual size. Resolving power is a measure of how clearly you can see the object. When you look at two lines on a page ( ), you can "resolve" the difference between the two with your naked eye. However if the lines are placed very close together at some point you would not be able to distinguish them as separate lines ( ). Magnification in a microscope can be increased indefinitely but at some point the magnification will exceed the resolving power of a light microscope and the image will appear blurry. The maximum resolution (of ~0.2µm) in a light microscope is ultimately limited by the properties of light waves. Functionally, a light microscope can magnify about 1000 times, allowing us to see a small bacterium. Many objects, such as most organelles within a cell, are too small for the resolution of our light microscopes. To see these objects scientists need to use a different type of microscope, such as an electron microscope. Resolving power. There are many ways to increase the resolution of an object: 1) increase the size of the object (change magnification) by using different lenses or objectives 2) adjust the focus with the coarse and fine focus adjustment knobs 3) adjust the amount of light by adjusting the rheostat and the iris diaphragm 4) adjust depth of field (how many levels are in focus at a time) with the iris diaphragm 5) cleaning the slides and lenses Microscopes allow you to increase the resolution by all of these means. The more detail you can distinguish, the greater the resolving power (or resolution). We will use two different types of light microscopes in these labs: compound microscopes and dissecting microscopes. Compound microscopes are capable of greater magnification and resolution, but need to be used with very small objects that the light can easily pass through. Dissecting microscopes are used for examining larger, thicker objects at lower magnification. GOALS AND OBJECTIVES FOR MICROSCOPE PORTION OF LAB 1. Be able to safely and effectively use the microscope. 2. Recognize and name the parts of a compound microscope and their functions. 3. Recognize and name the parts of a dissecting microscope and know their functions. 4. Focus a microscope slide at low, medium and high powers. 5. Correctly set up and put away a compound microscope. 6. Make a wet mount slide. 7. State the relationship between resolving power and magnification. 8. Calculate total magnification of a specimen on a microscope. Be able to estimate size. 9. Understand the relative sizes that correspond to units of the metric system. 10. Recognize the organisms you observe. Know their classification and characteristics. Microscopes Page 1

2 Metric System In order to understand magnification and resolution, you must be comfortable with metric units of measurement. Here is a conversion chart (learn it!): Units meter centimeters millimeters micrometers nanometers 1 meter m 1 m 100 cm 1000 mm 1,000,000µm 1,000,000,000nm 1 centimeter cm 1/100 m 1 cm 10 mm 10,000 µm 10,000,000 nm 1 millimeter mm 1/1000m 1/10 cm 1 mm 1000 µm 1,000,000 nm 1 micrometer µm 10-6 m 10-4 cm 1/1000 mm 1 µm 1000 nm 1 nanometer nm 10-9 m 10-7 cm 10-6 mm 1/1000 µm 1 nm 10cm 100 mm 100,000 µm 100,000,000,nm 1.Obtain a small metric ruler. Take out the ruler and sketch a centimeter, a millimeter and (attempt) a micrometer. centimeter millimeter micrometer 2. Select the appropriate metric units and estimate the following measurements: 1.the length of your index finger 2.the thickness of a microscope slide 3.the thickness of a coverslip (think about how to measure this accurately) 3. Using your book, find the approximate length of these objects using the appropriate units: 1. a plant cell 2. an animal cell 3. a mitochondrion 4. a bacterial cell 5. a virus 6. a protein (depends on book) Microscopes Page 2

3 COMPOUND MICROSCOPE You will be assigned a microscope that you will use for the rest of the semester. A sign up sheet will go around. Write down your name and the microscope number. You will be responsible for the condition and care of that microscope. Use of the Microscope Refer to the drawing of the microscope on the opposite page. 1. Plug in the microscope. 2. Use the lens paper in your desk drawer and clean the glass surfaces of the microscope. ALWAYS USE LENS PAPER TO CLEAN GLASS SURFACES ON THE MICROSCOPE. NEVER USE PAPER TOWEL OR KIMWIPES. 3. Turn on the light source to the highest intensity. 4. Find the iris diaphragm and open it to its widest position. 5. Make sure that the lowest objective (4X) on the revolving nosepiece is directly over the stage. ALWAYS BEGIN WITH THE LOWEST POWER OBJECTIVE WHEN OBSERVING SPECIMENS. 6. Find the coarse focus knob and turn the knob until the stage is at its lowest position. 7. Take the letter "e" slide and clean it with a Kim wipe tissue. Make sure not to get fingerprints on the glass. 8. Pull the stage clip back and place the slide in the slide holder. 9. Use the slide holder adjustment knobs to move the letter "e" over the light source. 10. Use the coarse focus knob and raise the stage until it is in its highest position. Then, slowly lower the stage until the letter "e" comes into focus. ALWAYS BEGIN OBSERVING A SPECIMEN WITH THE STAGE IN THE HIGHEST POSITION. 11. Adjust the iris diaphragm until the amount of light is comfortable for you. 12. Use the fine focus knob and adjust until the letter "e" comes completely into focus. 13. Adjust the distance between the eyepieces until you can see one image with both eyes. 14. On one eyepiece is the diopter ring. Close that eye and look through the microscope with the other eye. Adjust the fine focus knob. Now switch eyes and adjust the DIOPTER RING to get the other eye into focus. You have adjusted for the difference in the vision of each eye. 4

15. Center the letter "e" in the field of view. 16. Change the objective to medium power (10X) while looking from the side. DO NOT ADJUST THE FOCUS KNOBS TO LOWER THE STAGE.

4 15. Center the letter "e" in the field of view. 16. Change the objective to medium power (10X) while looking from the side. DO NOT ADJUST THE FOCUS KNOBS TO LOWER THE STAGE. You will need to adjust the fine focus slightly to bring it back into sharp focus. ALWAYS LOOK FROM THE SIDE WHEN CHANGING THE LENS IF YOU NEED TO MAKE A LARGE ADJUSTMENT TO THE FOCUS, YOU NEED TO GO BACK TO 4X AND GET THE OBJECT BACK IN FOCUS. 17. To change to high power (40X), do the same as in #15 and #16. Be sure to look from the side to make sure the lens doesn t hit the slide. NEVER USE THE COARSE FOCUS KNOB WHEN USING HIGH POWER! 18. You will need to adjust the amount of light at different magnifications. Use the iris diaphragm and light intensity to do so. 19. Oil immersion is difficult to use and requires a drop of oil. We will not use the oil immersion objective in this class.

5 Estimation of Size and Resolution (Letter e ) You can determine the size of the specimen in the microscope by comparing it to the width of the field of view. Here are the sizes of each field of view: Objective Magnification Eyepiece Magnification Total Magnification Diameter Of The Field Of View Scanning 4X 10X 40X 4600 µm Medium 10X 10X 100X 1800 µm High 40X 10X 400X 460 µm For the medium power objective the diameter of the field of view is 1800 µm wide. If I am looking at a specimen that can fit 2 times across the field of view, then I divide 1800 by /2 = 900 µm. 10X Objective 1800 µm How wide is the letter "e" on your slide? Write down your calculation. Resolution Look at the letter e on high power (40x). Turn the light source all the way up. Open and close the iris diaphragm to determine the position that allows you to see the most detail (maximum resolution) of the fine structure of the paper and ink. What adjustment of the iris diaphragm gives the maximum resolution? Note that the best adjustment depends on the specimen and how transparent and thick it is. In addition to adjusting resolution, the iris diaphragm adjusts depth of focus. Depth of focus is the thickness of the specimen that can be in focus at one time.

6 Observation 4 Review of Microscope Use 1. How should the microscope be lifted and carried? 2. A microscope should not be slid across the table top. Explain the reason for this rule. 3. What are the basic rules for use of the microscope (name at least 5, not including the above)? 4. Name five ways to increase resolving power of your microscope. 5. Why don't we allow you to use the coarse focus knob while on high power?

7 POND WATER OBSERVATIONS INTRODUCTION Pond water is a rich media for growth of microorganisms. Holding pond water up to the light you may notice a few tiny organisms moving about. More often it appears cloudy or greenish in color. Under the microscope, the faint hint of living creatures becomes a miniature world of activity, a community of living things. Imagine the shock of discovery when the early microscopists looked at a drop of pond water beneath a microscope. Nothing in their worldview had prepared them for what they were about to discover. The diversity of organisms that occupy pond water is amazing. The dominant life forms are primarily protists (which are mostly single-celled organisms) and bacteria. Other aquatic organisms include a host of multicellular animals: roundworms, flatworms, segmented worms, rotifers, aquatic insect and insect larvae, crustaceans, and more. Within a small volume of pond water an entire food web can exist. The base of the food web are the various producers. These include a variety of photosynthetic organisms, including both bacteria and protists. Photosynthetic pigments in these organisms often make them look green, yellow-green, or yellow-brown in color. The primary producers are food for numerous heterotrophs that you may see scurrying about in search of food. The size of these varies from a few hundred micrometers to large predatory insects that can easily be seen with the eye. GOALS AND OBJECTIVES 1. Recognize and distinguish various phyla of organisms under the microscope. 2. Draw distinguishing features of organisms and estimate their approximate size. 3. Use a simple key to identify the phyla of pond water organisms. 4. Distinguish between characteristics of living organisms and debris or an air bubble. PROCEDURE 1. Use a dropper to obtain a sample of pond water. Depending on the size of the organism you wish to observe choose either a deep-well or a flat slide (flat slides are easier to observe and therefore better in most cases). Cover all specimens with a coverslip. 2. Begin observing your specimen with the 4x objective. Look around to see what types of organisms are in your water. 3. Some will move too quickly to see clearly. Be sure to distinguish between a living organism and debris or an air bubble. 4. Once you see an interesting organism or object increase the power to 10x, then 40x objectives. If you have a rich culture with interesting organisms you may wish to share it with the class. 5. Once you have identified an interesting organism carefully draw it showing as much of the organism s detail as you can, especially those features that helped you with your identification. Try to estimate the size of the organism

Drawing Instructions 1. Make each drawing inside a circle representing the field of view you observed under the microscope. 2. Draw the organism as large as necessary to show detailed features. 3.

8 Drawing Instructions 1. Make each drawing inside a circle representing the field of view you observed under the microscope. 2. Draw the organism as large as necessary to show detailed features. 3. Draw a size bar to indicate the proportional size of the specimen in the field of view (the size it appeared when looking through the microscope). 4. Include the name of the phylum of the organism you drew. 5. Label each drawing with the lens and the total magnification used to make your drawing. 6. Estimate the size of the organisms using the technique learned in the first microscope lab. A summary of technique may be found at the back of the handbook. Be sure to include the equation you used to find the size and a bar line to indicate which direction the measurement was taken. 7. Include the location where the sample was collected. This should include the type of source, a location or address and the city. Example of a pond water drawing: Phylum: Objective Lens: Total Magnification: Estimated Size: Arthropods 10x 100x length ~720µm (Indicate length or width and units) Location collected: Copeland Creek Sonoma State Univ., Rohnert Park Comments: wiggles around from side-to-side Size Calculation: 1800µm = 720µm 2.5 Magnification and Diameter of the Field of View objective x eyepiece = Total magnification diameter of the field of view scanning objective 4 x 10 = 40x 4600µm medium power objective 10 x 10 = 100x 1800µm high dry 40 x 10 = 400x 460µm

9 DISSECTING MICROSCOPE Dissecting microscopes are designed to examine larger specimens and will be used when we look at animals and plants later in the semester. They are used in a slightly different manner than the compound microscope. Using the Dissecting Microscope A. The dial on the top of the dissecting microscope adjusts the amount of magnification. The dial shows the total magnification. 1. What is the lowest total magnification for the dissecting microscope? The highest? What are the lowest and highest magnifications for the compound microscope? B. Plug in the microscope. C. Find the switch and turn on the light source. D. Play with the light switch, until you can turn on just the lower light source, just the upper light source, and then both light sources. E. Separate the eye pieces until they match the distance between your eyes. F. Turn the magnification knob to the lowest magnification. G. Place your finger on the stage, and use the focus knob to bring your fingernail into focus. H. Use the magnification knob to increase the magnification as much as possible while still retaining the resolution. I. On the side benches you will find specimens to observe under the dissecting microscope. Observe several objects with this scope. J. When you are done, lower the microscope head to its lowest position, and turn the light off. Comparison between the Dissecting and Compound Microscopes 1. What is the advantage of using a dissecting microscope? 2. What is the advantage of using the compound microscope? 3. Make a list of objects that can only be viewed with a dissecting microscope (not a compound microscope). 4. What specimens seen previously in the lab would not be good to view with the dissecting microscope?

MICROSCOPE LAB. Resolving Power How well specimen detail is preserved during the magnifying process.

MICROSCOPE LAB. Resolving Power How well specimen detail is preserved during the magnifying process. 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