الرئيسية  |  القسم الاكاديمي  |  القسم الاعلامي  |  اللوحة الافتراضية  |  القسم الاخباري  |  نظام المحاضرات  |  قسم المعلومات

انت هنا الان : شبكة جامعة بابل > موقع الكلية > نظام التعليم الالكتروني > مشاهدة المحاضرة

microscope

الكلية كلية هندسة المواد     القسم قسم البوليمرات والصناعات البتروكيمياوية     المرحلة 4
أستاذ المادة شاكر جاهل ادريس المحنا       6/8/2011 9:10:38 AM
  2. INTRODUCTION TO THE MICROSCOPE
  3. LAB A
  4. Our goal is to become familiar with the wide range of cell types using the best tool for
  5. directly observing cells, the microscope. First we will learn to use the microscope well and then
  6. we will look at a variety of cells, both living and unstained, and fixed and stained.
  7. I. USING THE LIGHT MICROSCOPE
  8. Introduction
  9. One of the most important tools used by biologists is the microscope. It is an exquisitely
  10. sensitive arrangement of lenses that, when used properly, permits us to examine living and fixed
  11. material that we would not be able to see otherwise. A light microscope, by virtue of its lens
  12. system, extends our vision a thousand times, so that objects as small as 0.1 µm (10-7 meters) in
  13. diameter can be seen. Since average cells range in diameter from 1 µm (bacteria) to about 30 µm
  14. (epithelial cells), the microscope is a perfect tool for directly observing cells. Although three
  15. hundred years have passed since its invention, the standard light microscopes of today are based
  16. on the same principles of optics as microscopes in the past.
  17. Microscope quality depends upon the capacity to resolve, not magnify, objects.
  18. Resolution is the ability of the microscope to distinguish detail. Merely magnifying an object,
  19. without increasing the amount of detail seen, is of little value to the observer. Modern
  20. microscopes increase both magnification and resolution, simultaneously, by matching the
  21. properties of the light source and precision lens components.
  22. Even with sufficient magnification and resolution, a specimen can only be seen on a
  23. microscope slide if there is sufficient contrast among its parts. Contrast is based on the
  24. differential absorption of light by these different parts. Often a specimen will consist of opaque
  25. parts or will contain natural pigments, such as chlorophyll. Cells or subcellular structures
  26. containing pigments are visible, but what happens with the majority of biological materials that
  27. are highly translucent structures or very thin sections of specimens? The microscopist may
  28. improve contrast by the use of stains or dyes that bind to cellular structures and chemicals or
  29. may elect to use nonspecific stains which stain all structures. The study of staining is a field in
  30. itself called cytochemistry or histochemistry.
  31. Other types of microscopy allow us to probe cells and their organelles in different ways.
  32. Consult your text to develop an understanding of transmission electron microscopy, scanning
  33. electron microscopy and atomic force microscopy (pp. 86-89).
  34. Materials:
  35. binocular compound microscope
  36. lens paper
  37. prepared slide with crossed threads
  38. ruled stage micrometer (2 mm with 0.01 and 0.1 mm divisions)
  39. Methods:
  40. Examining and adjusting the compound microscope
  41. Before you begin, check to make sure that your microscope is ready to use. It should be
  42. plugged in with the light source off and the rheostat set at 1 or the lowest number. The shortest
  43. objective (lowest magnification power) should be in place and the stage lowered. Make any
  44. necessary corrections before turning on the light source and increasing the light intensity.
  45. 1. The oculars are the lenses you look through. The oculars can be adjusted to match the
  46. distance between the eyes of different observers (interpupillary adjustment). Should you wear
  47. your eyeglasses when using a microscope? The answer to this question is qualified. If you are
  48. nearsighted or farsighted, you should not wear your glasses for microscopic observations as the
  49. adjustments made in focusing the microscope compensate for these eye problems. You should,
  50. however, wear your glasses if you have astigmatism, since this problem is not corrected by the
  51. lenses of the microscope. Note the magnification stamped on the housing of the oculars on your
  52. microscope.
  53. 2. Attached to a rotating nosepiece, or turret, at the base of the body tube are a group of three or
  54. four objectives. Rotate the nosepiece and notice that a click is heard as each objective comes
  55. into position. The objective gathers light from the specimen and projects it into the body tube
  56. and through the oculars. Each objective has numbers stamped on it. One of these numbers
  57. identifies the magnification of the objective. Note the magnifications on each of the objectives
  58. of your microscope.
  59. 3. The total magnification is calculated by multiplying the magnifications of the ocular and
  60. objective lenses on the microscope being used. Calculate the total magnification for each ocularobjective
  61. combination on your microscope. How large would a 30 µm object appear if it were
  62. magnified 40-fold? 100 fold?
  63. 4. Light travels from the objectives through a series of magnifying lenses in the body tube to the
  64. ocular. In some microscopes, the body tube is straight. In others, the oculars are held at an
  65. angle, and the body tube contains a prism that bends the light rays so that they will pass through
  66. the oculars.
  67. 5. The surface or platform on which you place the microscope slide is the stage. Note the
  68. opening (stage aperture) in the center of the stage. On some microscopes, the stage is movable
  69. and is called a mechanical stage. Movement is controlled by two knobs located on the top, side
  70. or bottom of the stage.
  71. 6. The area under the stage, called the substage, may contain a diaphragm, a condenser, or both.
  72. a. Diaphragm--The diaphragm regulates the amount of light passing from the light
  73. source through the specimen and through the lens system of the microscope. By properly
  74. adjusting the diaphragm, you can provide better contrast between the surrounding
  75. medium and your specimen, thus greatly improving your image of the specimen.
  76. b. Condenser--The condenser consists of a series of lenses that focus light on the
  77. specimen. It is moved up and down by a knob at its side. By properly adjusting the
  78. condenser, you can greatly improve the clarity of the specimen.
  79. 7. Your microscope has a built-in illuminator. It is built into the base of the microscope and
  80. controlled by an on/off switch. You can control light intensity by adjusting the voltage of a
  81. transformer attached to the illuminator. Use low or medium settings for most microscopic
  82. observations.
  83. 8. Return the microscope to its "ready" position, i.e., put the 4X objective in place, lower the
  84. stage, turn down the illuminator to its lowest setting, and turn off the illuminator power. You
  85. may also wish to unplug the cord and wrap it around the base; then cover the microscope if a
  86. cover is available.
  87. Focusing your microscope
  88. 1. If necessary, clean the oculars and objectives using lens paper. Do NOT use Kim-wipes,
  89. facial tissue, paper towels, or your sleeve! (These materials all tend to scratch these highprecision
  90. lenses.)
  91. 2. Obtain a slide with a set of three crossed threads. Examine it with your eyes before placing it
  92. on the microscope stage. Now move the slide so that the area with the threads is located over the
  93. stage aperture. The 4X objective should be in place and the microscope stage down.
  94. 3. Turn on the illuminator, raise the light intensity to a comfortable brightness while looking
  95. through the eyepieces. Open the diaphragm halfway. Position the condenser as high as it will
  96. go. Readjust the light as needed.
  97. 4. Center the specimen into the field of view by using the mechanical stage.
  98. 5. Raise the stage (or lower the objective) using the coarse focus knob until the first thread
  99. comes into reasonable focus. Look and note how close the objective is to the slide. To adjust
  100. the focus for your right eye, close your left eye and use the fine focus knob to bring the middle
  101. thread into optimal focus. Now, close your right eye and adjust the left ocular lens until the same
  102. thread is in focus. Open both eyes. You should now have a well-focused binocular view of the
  103. threads.
  104. 6. Switch from the 4X to the 10X objective by turning the nosepiece and watching from the side
  105. as you move it into place. Do NOT hold the objectives while turning; or you are likely to
  106. unscrew them and have them fall out or become misaligned. The objective should click into the
  107. proper position.
  108. 7. Re-center the specimen while looking through the oculars using the stage adjustment knobs.
  109. (Locate them with your eyes and hands before looking through the microscope.)
  110. 8. Adjust the focus using the fine adjustment knob if necessary. Adjust the light as needed.
  111. 9. Adjust the condenser to increase the contrast of the image by moving the condenser as close
  112. to the stage and slide as possible and then move it back down approximately half a centimeter.
  113. You will want to play with this adjustment to optimize the lighting for each specimen and
  114. magnification.
  115. 10. While looking from the side, switch to the 40X objective by turning the nosepiece. Avoid
  116. hitting the slide with the objective.
  117. 11. Adjust the focus (fine adjustment knob only!), the light and condenser as needed to optimize
  118. your view.
  119. Determining the depth of field
  120. Like the human eye, the lenses of your microscope provide a limited depth of field. This
  121. means that only part of the object will be in sharp focus while areas above and below that part
  122. will be slightly out of focus or not in focus at all.
  123. 1. Go back to the 4X objective and examine the three threads together. Adjust the fine focus
  124. knob and note as the three threads in turn come into focus. Which thread is on top, which is in
  125. the middle, and which is on the bottom?
  126. 2. Repeat this examination using the 10X and 40X objectives. Which objective has the greatest
  127. depth of field?
  128. 3. Given that each thread is about 0.3 mm thick, what do you estimate the depth of field to be for
  129. each magnification?
  130. Measuring the field of view
  131. 1. The ruled stage micrometer that you have is a tiny ruler. It is 2 mm long with 0.1 and 0.01
  132. mm divisions. Put your microscope in the ready position with the 4X objective in place and the
  133. stage micrometer slide on the stage with the ruling centered over the aperture and under the
  134. objective lens.
  135. 2. Focus on the rulings using the coarse and fine focus knobs. Use the rulings to determine the
  136. width of the field of view. Record this value in both mm and µm. Draw a picture indicating
  137. how large something 100 µm in diameter would appear in this field of view.
  138. 3. Repeat these measurements and drawings using the 10X and 40X objectives.
المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .