BIOLOGY 163 LABORATORY USE OF THE COMPOUND LIGHT MICROSCOPE (Revised Fall 2012) Microscopes in various forms are important tools for biologists. Two types of microscopes that you will use frequently are the compound and dissecting light microscopes. Of these two, the compound microscope is more difficult to use well. Successful use of the compound light microscope depends on a variety of factors including quality of slide preparation, proper focusing, and adjustment for optimal illumination. Compound light microscopes employ a series of lenses to enlarge (i.e., magnify) the image of the object being studied. Total magnification is the product of the magnifying power of the objective lens times the magnifying power of the ocular lens. Typical microscopes have four objective lenses (4X, 10X, 40X, and 100X) that can be rotated into position. Combined with a 10X ocular lens (standard on our microscopes), this gives the user the option of 40X, 100X, 400X and 1000X magnification. Magnification, however, is only part of the equation. The real purpose of a microscope lies in its ability to allow the user to see small details (i.e., resolving power). Resolving power is expressed as the limit of resolution (lr). This is the smallest distance by which two neighboring points can be separated and still be seen as separate entities. The limit of resolution of your naked eye is about 0.2 mm. That of a good quality light microscope is about 0.2 µm (1 µm = 0.001 mm), a 500-1000 fold improvement over your eye. Good illumination is critical to realizing the full potential of the microscope. In 1893, August Kohler described a method to achieve the most uniform illumination from a non-uniform light source such as a light bulb. The result has become known as Kohler illumination and is obtained (1) by focusing the bulb filaments at the plane of the condenser diaphragm and (2) by focusing an image of the lamp diaphragm at the plane of the object. In microscopes with built-in illuminators the microscope manufacturer has done Step (1). You will need to do step (2) after some initial set-up procedures with the microscope. You will use microscopes to study both "wet" and "dry" preparations. The latter are prepared for you and consist of thin sections (about 0.01 mm thick) permanently set in some mounting medium. You make "wet" preparations to examine fresh material. Information on preparing wet-mount slides is provided for your reference in Appendix B. USING A LIGHT MICROSCOPE WITH KOHLER ILLUMINATION Please respect the equipment by observing the following precautions! Carry the microscope two hands: one supporting the arm and the other under the base. Keep the microscope UPRIGHT or the ocular lenses may fall out. If the lenses are dirty, clean them ONLY WITH LENS PAPER. Other substances may scratch the precision lenses. If you dirty a lens, clean it IMMEDIATELY with lens paper. ALWAYS focus by moving the stage/specimen AWAY from the lens. Failure to do so may allow the slide to contact the lens, potentially damaging both. ALWAYS focus on your specimen with a lower power before moving to a higher power. If you canʼt find your specimen at 100X, you will definitely not find it at 400X (and you may risk damaging the specimen or the microscope if you try). ALWAYS use a cover slip when observing a wet mount specimen. 1
Use the following procedure to properly set up the microscope for viewing specimens. Refer to Appendix F if you are unfamiliar with the parts of the microscope! 1. Using the condenser height adjustment knob, place the condenser in its uppermost position. Open the condenser diaphragm fully by sliding the lever on the front of the condenser all the way to the left. 2. Turn on the lamp using the power switch located on the front of the microscope base. Open the lamp diaphragm fully by rotating the ring on the top of the lamp clockwise until it stops. You can adjust the intensity of the light by rotating the numbered ring on the right-hand side of the base. 3. Rotate the objective lenses until the 10X objective locks into place over the condenser. 4. Open the spring-loaded clip on the stage and insert your slide. Use the stage controls to position your specimen in the center of the beam of light. Do this looking from the side, with your naked eye; donʼt attempt to hunt for the specimen while looking through the ocular lenses! 5. Raise the stage so that the specimen is close to the objective lens. Look through the oculars and slowly lower the stage with the course focus adjustment knob until the specimen become visible. Once the specimen is visible, use the fine focus adjustment knob to make the specimen as clear as possible. Remember to ALWAYS focus by moving the stage/specimen AWAY from objective lens! 6. Adjust the distance between the ocular lenses. WITH BOTH EYES OPEN, you should see a single, fully illuminated, circular field of view. (TIP: If you are having difficulty with this step, your eyes may be too close to the ocular lenses!) 7. Adjust the focus to accommodate for differences between your two eyes. Close your left eye. Looking with your RIGHT EYE through the RIGHT OCULAR, focus the image using the FINE FOCUS ADJUSTMENT KNOB. Close your right eye. Looking with your LEFT EYE through the LEFT OCULAR, focus the image using the DIOPTER ADJUSTMENT RING on the base of the left ocular (do NOT touch the fine focus adjustment knob). Open both eyes. 8. Adjust the condenser to focus the light on the specimen. Rotate the lamp diaphragm ring counterclockwise until the diaphragm is at its minimum opening. A bright spot with blurry edges should be visible in the center of the field of view. (If the spot is not centered, ask your instructor how to adjust it.) Using the condenser height adjustment knob, slowly lower the condenser until the edge of the spot comes into focus. (TIP: You shouldnʼt need to move the condenser very far from its uppermost position.) Rotate the lamp diaphragm ring clockwise until the light just fully illuminates the filed of view. 9. Close the condenser diaphragm by sliding the lever on the front of the condenser toward the right. Adjust this to obtain optimal contrast for your specimen. Always adjust contrast by using the condenser diaphragm or the lamp intensity control. DO NOT adjust the height of the condenser, as this changes the focus of the light on the specimen! 10. If you wish to increase the magnification of the image, carefully rotate the 40X objective into place WITHOUT lowering the stage. You should now be able to bring the image into focus using ONLY the fine focus adjustment knob. In order to optimize the light, repeat steps 8 and 9 with the new objective. Avoid rotating the course focus adjustment knob, as this may cause the slide to impact the lens, potentially damaging both! NOTE: SEE APPENDIX C BEFORE ATTEMPTING TO USE THE 100X OBJECTIVE LENS! 2
APPENDIX A Use of the Phase-Contrast Microscope Phase-contrast microscopy allows the viewing of faint or lightly colored specimens without the use of stains. The phase ring, located just beneath the stage of the microscope, adjusts the light passing through specimen such that contrast between the specimen and the background is enhanced. Use of the phase-contrast microscope is very similar to use of the standard compound light microscope. However, in order for the phase-contrast properties of the microscope to be realized, the number on the phase ring must match the power of the objective being used (10X and 40X in most cases). Be sure to rotate the phase ring to the proper setting each time you change the objective. When using the phase ring, you do not need to make adjustments to the condenser diaphragm. Do not touch any knobs or controls with which you are unfamiliar--doing so could put the phase ring out of adjustment and the phase-contrast properties of the microscope will be reduced. If you are not certain of how to use the microscope properly, please ask your instructor for assistance. APPENDIX B Preparation of Wet-Mount Slides Wet-mount slides are made when you wish to view fresh specimens (as opposed to those that have been stained, fixed, and sectioned, as is the case with prepared slides). Make wet preparations by placing a small drop of water or other fluid in the center of a clean slide. Center the object of interest in the drop of liquid. Lower a clean cover slip over the drop gently to avoid trapping air bubbles. If done properly, the moisture of the drop will completely fill the space under the cover slip, with no excess outside that might run onto the microscope stage. If the coverslip appears to be "floating" on top the slide, you have used too much liquid! You will either need to make a new slide or wick some of the liquid away with a Kim- Wipe or paper towel. Studying wet preparations should show you that a microscopic preparation, while thin, does have appreciable thickness when observed with the microscope. A sense of the depth of the field is an important feature of study with the microscope. You can see depth of view by using the fine adjustment. As you focus up and down, the two-dimensional images at various levels become integrated in your mind to form a notion of the third dimension of the preparation. APPENDIX C Use of the Oil Immersion Lens Since oil has a higher refractive index than air, viewing a specimen through a drop of can increase the resolving power of the microscope at high magnification. The 4X, 10X and 40X objectives are dry objectives they are designed to be used in air. However, the 100X objective is a lens specially designed to be used with oil. To use this lens: 1. Rotate the 40X objective out of the way. Place a drop of immersion oil directly over the area on the slide you wish to observe. Rotate the oil immersion objective (usually 100x) into position. When doing this, watch carefully from the side of the microscope so that the objective does not contact the slide. 2. Focus the specimen using the fine focus adjustment knob and adjust for proper Kohler illumination. 3. When finished, WIPE ALL OIL FROM THE OBJECTIVE WITH LENS PAPER. Clean up any oil that may have found its way to other lenses or parts of the microscope! 3
APPENDIX D Use of the Ocular Micrometer One ocular lens of your microscope should contain a glass disk with fine divisions that are numbered. This is an ocular micrometer--it is used to measure objects in the field of view (Figure 1). 0 10 20 30 40 50 Figure 1. Scale of an ocular micrometer It is important to note that the numbers on the ocular micrometer DO NOT represent any fixed unit of distance! When you increase magnification, the field of view (i.e., the total area visible through the microscope) decreases, and the ocular micrometer becomes superimposed over a smaller area. As a result, the physical distance represented by each small division (or ocular unit ) decreases with each increase in magnification. For this reason, you need to calibrate the ocular micrometer for the specific microscope you are using! The ocular micrometer is calibrated with a stage micrometer, a precise scale prepared as a dry mount slide. Since the units on the stage micrometer are constant, it may be used to measure the ocular micrometer when used with the different objective lenses. This procedure has already been done for the ocular micrometers you will be using, and the results are provided in Table 1. You will need to use this information to be able to accurately measure the specimens that you view! Table 1. Ocular unit distances of ocular micrometers used at Colby College. One ocular unit is defined as the smallest division of the ocular micrometer. At this magnification: 40X (4X objective) 100X (10X objective) 400X (40X objective) 1000X (100X objective) Each ocular unit equals: 0.025mm (25um) 0.010mm (10µm) 0.0025mm (2.5µm) 0.0010mm (1.0µm) The ocular micrometer is a useful tool for measuring microscopic specimens, but the micrometer itself is not typically shown in illustrations or micrographs. For these applications, a scale bar should be used (Figure 2). Itʼs helpful to draw the scale bar approximately the same length as the specimen of interest, with the actual distance being determined using the ocular micrometer and the information in Table 1. 5µm Field of View Specimen Scale Bar Figure 2. Illustration of a specimen with a scale bar included. 4
APPENDIX E Understanding the Limit of Resolution The limit of resolution (lr) depends upon several factors; it is calculated using Abbe's equation: lr = 0.61 λ n sin α where: λ = the wavelength of illumination used (Fig. 1) n = the refractive index of the medium used between the objective lens and the specimen. n (air) = 1; n (oil) = 1.56 α = half the angle of light entering the objective lens (Fig.2); the theoretical maximum value of sin α is 1. Notice that the limit of resolution becomes smaller (resolving power increases) as the numerator becomes smaller and/or the denominator becomes larger. With most compound light microscopes, you can manipulate the size the angle α (by adjusting the condenser diaphragm) and the value of n (by using an oil immersion lens). The wavelength of light is fixed unless you add a colored filter to the light source. UV Violet Blue Gr een Yellow Or ange Red IR 4000 A 4100 4700 5200 5800 6000 6500 7000 A Figure 1. The Visible Light Spectrum Objective lens Slide Cone angle = 2! Stage Condenser lenses Condenser iris diaphragm control 2!! Figure 2. The angle of light entering the objective lens. 5
APPENDIX F The Olympus CH-2 Compound Light Microscope 6