ch18im - CHAPTER 18:TECHNIQUES IN CELL AND MOLECULAR...

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C HAPTER   18:T ECHNIQUES  I N  C ELL  A ND  M OLECULAR   B IOLOGY The Light Microscope: Principles I. Components of the light microscope – a microscope produces an enlarged image of an object A. Light source - external to microscope or built into base; illuminates specimen B. Substage condenser lens - gathers diffuse rays from light source; illuminates specimen with a small cone of bright light intense enough to allow very small parts of a specimen to be seen after magnification C. Objective lens – collects light rays focused on specimen by condenser lens; collects 2 kinds of light rays passing through slide/specimen 1. Rays altered by specimen – emanates from the many parts of the specimen; focused by objective lens in scope column forming real, enlarged image of the object 2. Rays not altered by specimen - pass directly into objective; form visual field background light D. Ocular lens - image formed by objective lens is used as an object by ocular lens (a second lens system); E. Eye lens system - uses virtual image formed by ocular as an object to form a real image on the retina F. Focusing knob – when turned, knob alters relative distance between specimen & objective, focusing the final image precisely on plane of retina II. III. Resolution the ability to see 2 neighboring points in a field as distinct entities A. If an image is magnified beyond ability to resolve (see additional detail), result is empty magnification B. Degree of resolution defines objective lens quality; the extent to which fine detail in specimen can be discriminated or resolved C. Resolution attained by a microscope is limited by diffraction 1. Because of diffraction, light emanating from a point in specimen can never be seen as a point in the image, but only as a small disk 2. If the disks produced by 2 nearby points overlap, the points cannot be distinguished in the image 3. Thus, the resolving power of a microscope can be defined in terms of the ability to see 2 neighboring points in the visual field as 2 distinct entities 4. If 2 parts of a specimen are not separated by sufficient distance —> images merge (not resolved) D. Resolution is determined by the following equation & is limited by the wavelength of illumination & numerical aperture d = 0 .61 λ n s in α N . A . = n s in α 1. d = minimum distance that 2 points in specimen must be separated by in order to be resolved 2. λ = the wavelength of light (527 nm used for white light) 3. n 4. α = half the angle of cone of light entering objective lens (angle between normal to base of light cone & its side); α is a measure of light-gathering ability of lens; directly related to its aperture 5. N. A., the denominator of the equation, is a measure of the light-gathering qualities of the lens (a
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ch18im - CHAPTER 18:TECHNIQUES IN CELL AND MOLECULAR...

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