Appendix C Microscopy

Appendix C Microscopy - APPENDIX C: USE OF THE LIGHT...

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Unformatted text preview: APPENDIX C: USE OF THE LIGHT MICROSCOPE Much of your work in lab will depend on your ability to use the light microscope. This appendix is designed as a "hands on" guide. Before you begin following procedures outlined below, review the parts of the microscope and the path of light through it. Make sure you know the location and function of the parts discussed. Refer to the illustration at the end of this appendix. Microscopy: Some Fundamental Principles The three main features of microscopic imaging are: magni&ication, resolution, and contrast. These three criteria are fundamentally related to one another. Proper microscope technique takes into account the physical limitations of each aspect of imaging, as well as their relationships to each another. Successful microscopy is actually a practice in compromise; just because some detail in a microscopic image may not be visible does not mean that it is not there. Each component of microscopic imaging is given brief discussion below. Magni&ication. The magniGication of a lens or of a system of lenses, such as a microscope, is simply the factor by which the image is made larger than the original object. There is virtually no limit to the magniGication that can be achieved with any microscope. However, there is a real limit to the useful magniGication. Beyond a certain point the image appears 'fuzzy' and is said to have empty magniGication. The magniGication at which you are observing details through your microscope is computed simply as the product of the Gigures printed on the objective and ocular. For example: using a 40x objective with a 10x ocular gives an image magniGied 400 times. Resolution. The resolving power of a microscope lens is deGined as the ability of the lens to separate two tiny spots of light in a dark Gield (or two dark specs against a bright background) and render them as two spots rather than one. The resolution is the smallest such distance that can be detected. Factors that affect resolving power are: 1.) the light ­gathering power of an objective (called "angular aperture"); 2.) the refractive index of the medium (air vs. oil); 3.) the wavelength of the illuminating light source. The best possible resolution obtainable with a light microscope is 0.2 µm. Contrast. Contrast is the ability to compare in order to show unlikeness or differences. Think of it as the ability to discern microscopic detail from the background. Sometimes the ability to detect 1 and increase inherent contrast in the specimen is more important than the ability to resolve critical detail. In bright &ield microscopy, an object that is visible only because it absorbs light is called an amplitude object — such an object requires natural pigmentation, as in chloroplasts, or the use of stains, as is usually done with Gixed tissue. In phase contrast microscopy, an object that does not absorb, but rather bends, retards, or advances light waves in relation to others nearby is said to be a phase object (because it phase ­shifts light waves). The edges of the object deGlect light into regions of the lens system where light gets absorbed and phase shifted; undeGlected light gets absorbed. Many objects are mixed phase ­amplitude objects. We will exclusively use bright Gield microscopy. Care of the Microscope When moving a microscope always use two hands, one grasping the arm of the scope and the other placed beneath the base. Before you use the microscope, make sure all lenses of the objectives and oculars are clean; a blurred image is usually the result of a dirty objective, ocular or slide. Use lens paper (or Q ­tips) and breath for cleaning lenses. If you Gind you can't clean a lens satisfactorily please check with your lab instructor. Keep the stage clean and immediately wipe up any liquid that may fall on it. Keep your scope free of dust and store it with the low power objective in place. Getting Started The microscopes have a variable switch on the base of the scope; this controls the intensity of the illumination. Before plugging in the microscope, make sure that the switch is in the 'off' position. You may now turn on the illuminator: switch the knob on and turn it slightly clockwise to attain sufGicient illumination (not too much). If not already in place, bring the 10x objective in line with the condenser by rotating the revolving nosepiece that holds the objectives. Place a specimen slide on the microscope stage; be sure that the coverslip is on top. The spring ­loaded holder of the mechanical stage secures the specimen slide in place. Focus on the specimen with the coarse adjustment knob. To properly do this you must adjust the interocular distance (the distance between your pupils) and the diopter (eyepiece tube length), as outlined below. Adjusting interocular distance: 2 While looking through the eyepieces with both eyes, adjust the interocular distance by grasping the oculars and sliding them together or apart, whichever is required, so that perfect binocular vision is achieved. You'll also that notice that the distance from your eyes to the eyepiece also inGluences the Gield of view; position your eyes at the appropriate distance to allow full wide ­Gield viewing. Move your head back and forth slowly to facilitate this determination. Your eyelashes are greasy — if they touch the oculars, clean them off. Adjusting the eyepiece diopter: 1. Close the left eye and look at the image through the Gixed right eyepiece tube with the right eye. 2. Focus on the specimen with the coarse and Gine adjustment controls. 3. Close the right eye and look at the image through the adjustable left eyepiece tube with the left eye. 4. Focus on the specimen only by rotating the diopter adjustment ring located at the base of the left eyepiece tube. Do not touch the coarse or Gine adjustment controls while performing this operation. The specimen should now be in sharp focus with both eyes. Further adjustments Generally, these are the other adjustments of which you should be aware: • Position the height of the substage condenser with its height adjustment knob to just below its highest position. • Adjust the contrast: For examination of living cells and other objects of low contrast it may be desirable to close down the diameter of the condenser iris with its lever. This will increase contrast (at the expense of resolution). Examine the image while opening and closing the condenser iris. Usually it's best to set the condenser iris lever about half way open, so that the image is only somewhat dimmer than its brightest appearance. • Scanning, focusing, and controlling light intensity. One should scan the slide with one hand on the stage travel knob, while the other hand controls the Gine focus adjustment. You'll Gind that you'll be constantly focusing up and down through a specimen to see all the detail. This is because most biological specimens are thicker than the focal depth of the optical system (the distance between the upper and lower limits of sharpness in the formed image). Light intensity is controlled by the illumination knob on the base of the microscope. Do not control light intensity by moving the condenser or irises or it is likely you will end up with a poor image. 3 Note: This drawing may not depict the model that you will use. It is intended as a general schematic, with labeled parts. Your TF will acquaint you with the particulars of your instrument. Figure 1. Binocular Brightfield Microscope Parts 1. OCULAR (EYE-PIECE), 2. OBJECTIVE, 3. STAGE, 4. SLIDE HOLDER, 5. CONDENSER, 6. CONDENSER HEIGHT ADJUSTMENT KNOB 7. CONDENSER IRIS LEVER, 8. FINE FOCUS, 9. COARSE FOCUS, 10. FIELD IRIS DIAPHRAGM 4 ...
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This note was uploaded on 02/09/2012 for the course BIOLOGY 102 taught by Professor Anderson during the Spring '11 term at Harvard.

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