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Unformatted text preview: problem is that the small spot of illumination created by the condenser is also hot in addition to being bright. This heat may harm living specimens. To avoid such problems, you should make the required observations quickly, or lower the light intensity with the brightness control knob (do not lower the condenser). The condenser diaphragm regulates the diameter of the light beam passing through the condenser. As the condenser diaphragm is closed, specimen contrast increases. This phenomenon can be utilized to great advantage when observing specimens that have low inherent contrast such as living cells. You will have a chance to see this later. Be advised that microscopic resolution (see next section) is lost as the condenser diaphragm is closed. 3. The objective lenses. The function of the objective lens is to accomplish the first stage of magnification in the compound microscope. These lenses are threaded into the revolving nosepiece. NOTE: Remember that rotating the nosepiece should be done with the use of the “grooved” surface on the edge of the revolving nosepiece and not by pushing or tugging on the objectives themselves. Doing so could loosen the objectives and eliminate the parfocality of the microscope – this could result in ‘scope or slide damage. A further word of caution about the objective lenses concerns lens cleaning - improper cleaning of the objectives can result in a scratched lens. This kind of damage is prevented by keeping in mind that all Biology 05LA – Fall Quarter 2012 Lab 1 – page 4 exposed lens surfaces should be cleaned with lens paper only. DO NOT USE KLEENEX, LAB TISSUE, OR HANDKERCHIEFS! Your TA will demonstrate the best way to clean the lenses of your microscope. At this point you should note the markings on the three or four objectives on the nosepiece. These are shown in Figure 2. Two points need to be made here: First, the magnification listed gives the magnification achieved only with the objective lenses. Since the ocular lens, to be discussed later, magnifies the image formed by the objective lens ten times, the value for each objective lens magnification times the 10x magnification contributed by the ocular gives the total magnification for the combined (compound) lens system. Figure 2. Objectives of the Nikon Alphaphot. The Second, numerical aperture (NA) is a measure of the light gathering capacity of the objective lens. The greater the value of NA, the greater the resolving power of the lens. upper large numbers indicate the magnifying power of the objective; 4 - 100x. The number to the side or just below the mag. value indicates the numerical aperture of the objective; 0.1-1.25. The numbers at the bottom gives other information about the objective. Some optical principles involving the objective lens system. The resolving power of a lens system is defined as the minimum distance that two points can be spatially separated and still be seen as two individual points. The resolving power of the unaided human eye is 0.1 mm (100 m). That is, if two points are spaced closer than 100 m, the human eye sees them as a single point. The maximum resolving power of the light microscope is about 0.2 m. Since many cellular organelles are 1.0 m or less in length, they are at the limit of the resolving power of the light microscope. Depth of field for a given objective is a term that relates to the thickness of the microscopic field that is in usable focus at a particular time. This definition may be obscure at the moment, but the following exercise will help you to understand it. Before you begin, please refer to the content of #I. in the SUMMARY OF MICROSCOPE USE PROCEDURES on page 9. You will be expected to use the techniques described there for this and all subsequent microscopy exercises. a. Obtain a slide with three overlapping threads of different color. b. Using the protocol referred to above, obtain a low power (40x) image of the area where the threads overlap and then determine which of the three threads is on top, in the middle, and on the bottom. c. Now focus as best you can on the middle thread. Can you still see the top and bottom threads clearly? d. While still focused on the middle thread, bring the medium power objective into position and sharpen the focus on the middle thread. Can you still see clearly the top and bottom threads? e. Repeat step “d” using the high power objective. Are the top and bottom threads still visible? You should have seen that depth of field decreases as magnification increases. This relationship can be used by the microscopists in two ways: First, lenses with a thicker depth of field should be used when either locating smaller specimens or when you want to study the spatial Biology 05LA – Fall Quarter 2012 Lab 1 – page 5 relationships of larger structures where high resolution is not important. Lenses useful here are low power lenses. Second, lenses with a thinner depth of field should be used when you want to study the spatial relationships o...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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