Transcript
BACKGROUND INFORMATION ON THE STEREO DISSECTING MICROSCOPE AND CONVENTIONAL UPRIGHT MICROSCOPE The microscopes that we use in this laboratory are relatively inexpensive models of two of the great workhorses long used by investigators to study developmental biology. The stereo dissecting microscope is a binocular microscope that allows you to view your specimen from above the stage. The microscope design allows large objects, such as whole mounted objects, to be placed on the stage to be studied, which also allows for the object to be manipulated while magnified. Light can be added from within the microscope body to backlight the specimen or from above the stage to highlight surface features. The conventional upright microscope is the standard microscope that most people are already familiar with. This microscope is designed for high magnifications, which allows for a great deal of close analysis. Light is added through the body of the microscope to aid in the viewing of objects on the stage that are thin or transparent enough to transmit light directly through the specimen. General Care and Use of Microscope Lenses and Slides. These microscopes are delicate instruments, whose glass lenses and mirrors are fragile and difficult to replace. The slides and other objects we will view this semester are also delicate. By following a few simple guidelines we will be able to use them for years to come. Lenses should be cleaned only if absolutely necessary. Use fresh lens paper on oculars and objectives. Wipe the lens gently. Do not use a piece of lens paper more than once. Do not use any other material on lenses. The stage may be wiped off with a KimWipe. Wipe each slide before you put it on the stage. Slides collect a lot of debris. Wipe the slide thoroughly with a KimWipe. Hold the slide on the edges. Do not get fingerprints on a slide. Do not use water on a slide. Be sure that you wipe all oil off the slide and the lens after using 100x. PHYSICS, IMAGE RESOLUTION AND OBJECT SIZE The stereo dissecting microscope has both fixed magnification ocular lenses near the eyes and an adjustable objective lens near the object. The microscope has a very long focal length (the working distance between the object and the objective lens), which allows for large items on the stage, but the long focal length limits the magnification achievable in the objective lens. Thus, these microscopes are not useful for high magnification analyses. The physics of the microscope is dependent on unaltered or intensified bright white light, which consists of all wavelengths of the electromagnetic spectrum, reflecting off of the surface of the object on the stage. The glass lenses magnify the image of the
object on the stage and mirrors in the light path bring it to the eye. The working magnification, or resolution, is equal to that of the objective lens near the object, usually in a range of about 1X to about 4X, times that of the fixed 10X ocular lens near the eye. Maximum resolution is, therefore, 40X relative to the eye. The resolution capacity of the unaided eye is ~90um or 90,000nm. The conventional upright microscope, too, has fixed ocular lenses and adjustable objective lenses. However, the focal length of theses objective lenses is very short, thus allowing for high magnifications. These short focal lengths require the use of very thin material (usually sectioned material placed on microscope slides). The physics of these microscopes is dependent on intensified bright white light passing through thin cells or pieces of tissue on the stage. Again, glass lenses magnify the image from the stage and mirrors in the light path bring it to the eye. The working magnification, or resolution, is equal to that of the objective lens near the object, usually in a range of about 4X to about 100X, times that of the fixed 10X ocular lens near the eye. Maximum resolution is, therefore, 1000X relative to the eye To become more familiar you should examine the stereo dissecting microscope to learn the parts and their functions. a. Place a ruler in the same field of view as a penny and draw them together. Note the diameter on the drawing of the penny. 1. The lamp illuminates the object on the stage 2. Adjust the distance between the oculars to match the distance between your pupils 3. Use the adjustment knob (on the arm of the microscope) to bring the object into focus 4. The substage mirror aids in reflecting the light. Rotate the mirror between its two surfaces until the light is bright, non-glaring, and comfortable for your eyes 5. Adjust the oculars. The microscope has one fixed and one adjustable ocular. Cover the adjustable ocular and focus for the fixed ocular. Using both eyes, adjust the other ocular (by rotation) until focus is sharp for both eyes. 6. The knob (on the top or the side of the microscope) moves the lens system (magnification between 3x and 30x, depending on the microscope). Turn this knob and note the changing field of view and size of the object. To become more familiar you should examine the conventional light microscope to learn the parts and their functions. a. Place the slide on the stage. Fastenings hold the slide (by the edges). Turn the knobs at the side of the stage to move the slide. You will want to draw this preparation at several magnifications. 1. Turn on the substage illuminator. Bring the light to medium brightness
2. The oculars magnify the image 10x. Adjust for the distance between your eyes. 3. The nosepiece contains the objective lenses. Hold the nosepiece and put the 10x objective into position over the slide. (Do not turn the lenses by holding a lens.) 4. The focusing knobs are on the side of the arm. The coarse focus knob is used with the low power objectives. The fine focus knob is used for sharp focus and to focus the high power objectives. Bring an object on your slide into sharp focus. 5. Adjust the illumination. Turn the light up or down and fine-tune the condenser under the stage. 6. One ocular is fixed, and one is adjustable. Cover the fixed ocular and focus the 7. adjustable ocular to your eye 8. Turn the nosepiece and bring the 40x objective over the slide. Note that the object on the slide is nearly in focus. When you move from one objective lens to the next higher (or lower) power, the object on the slide should remain nearly in focus (parfocal). 9. Bring the object to sharp focus using the fine focus knob. b. Resolution and Contrast 1. Adjust the intensity of the illumination to a comfortable level 2. Adjust the contrast by racking the condenser up and down (use the substage knob) 3. Open and close the condenser diaphragm (using the lever projecting from condenser) to sharpen contrast. However, if the diaphragm is closed too far, resolution is lost. 4. Readjust the light intensity, if necessary 5. It may be necessary to readjust contrast each time magnification is changed. c. Precautions 1. After you place the slide on the stage, obtain initial focus using the 10x objective. 2. Use the 10x when locating objects on the slide. 3. Change to 40x after the object is centered at 10x. 4. Do not use the 40x (or 100x) lenses with thick preparations. You will probably crack the coverglass and could damage the lens. 5. Do not use the coarse focus knob with the 40x or 100x lenses. This knob moves the slide too great a distance.
