Microscopes: LM (Bright-field)

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Microscopes: LM (Bright-field)
2013-09-01 18:40:35
Cell BIO

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  1. Requirements
    • a.      a light source (external or internal) to illuminate the specimen
    • b.      substage condenser lens gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light that allows very small parts of the specimen to be seen after magnification
  2.                                                               i.      light rays then collected by __ which focus to form a real, enlarged image
    two types of light rays:
    objective lens

    • a.      those specimen alters
    •                                                                                                                                       i.      emanates from specimen parts and illuminates
    • b.      those it doesn’t: light from the condenser that passes directly into objective lens, forming background light of field
  3. 1.      the image formed by the objective lens is used as an __by a __, to form an enlarged and virtual image
    • object 
    • second lens system, the ocular lens
  4. Third Lens system
    1.      a third lens system located in the front part of the eye uses the virtual image produced by the ocular lens as an object to produce a real image on the retina
  5. total magnification
    empty magnification
    • a.      product of the magnifactions produced by the objective and ocular lens
    • b.      ability to see two neighboring points in the visual field as two distinct entities
    •                                                               i.      not being able to see even though you increased magnification
  6.                                                               i.      the more __that provide information about the image, the more __that can be seen

    Explain optical quality
    • photoreceptors 
    • detail 

    1.      the optical quality of an objective lens is measured by the extent to which the fine detail present in a specimen can be discriminated, or resolved
  7. Resolution is limited by __, which causes __.
    • diffraction
    • light emanating from a point in the specimen to never be seen as a point, but only a small disk
  8. What is the equation for limit of resolution
    •                                                               i.      : d= (.61λ/ nsinα) , where d is the minimum distance that two points in the specimen must be separated to be resolved,  is the wavelength of light (527 nm for white light), and n is the refractive index of the medium present between the specimen and objective lens.
    • 1.      α is equal to half the angle of cone of light entering the objective lens
    • a.      it is a measure of light-gathering ability of the lens and is directly related to its aperture
  9. In the equation for resolution, what is the denominator called?
    • 1.      the numerical aperture, which is a constant for each lens, a measure of its light gathering qualities
    • a.      for air, it is 1; for oil, it is 1.5
  10. Common rule for NA
    • a.      maximum useful magnification of a light microscope ranges between 500 and 1000 x the NA of the objective lens being used
    •                                                                                                                                       i.      anything higherà empty magnification
  11. High NA is achieved by using __.
    a.      lenses with a short focal length, which requires the lens to be placed very close to the specimen
  12. limit of resolution
                                                                  i.      If we substitute the minimum possible wavelength of illumination and the greatest possible NA= limit of resolution
  13. Limit of resolution of the LM and naked eye?
    • 1.      of LM: slightly less than 200 nm (can see large organelles, like nuclei and mitochondria)
    • 2.      of the naked eye: (has NA of 0.004) approx. 0.1 mm
  14.                                                               i.      resolving power also affected by __ [must be avoided by lensmakers]
    1.      lenses are not __
    • optical flaws, or aberrations (7) 
    • one, but many to correct for errors
  15. Visibility
    a.      concerned with actual observation (aka: contrast: difference in appearance between adjacent parts of an object or between an object and its background)
  16.                                                               i.      using a microscope, we place specimen between __ and view the light that is transmitted through the object (should be __or close to it)
    • light source and our eyes
    • transparent
  17. Staining
    problems with it
    •                                                               i.      absorbs SOME wavelengths; others are transmitted to eyeà colored specimen
    • 1.      different dyes bind to different biological molecules
    • 2.      problem: cannot be used with living cells
    • a.      they or the conditions are toxic
    • they may not penetrate the plasma membrane
  18.                                                               i.      Different types of LM use different types of __ 
    bright-field microscope
    • illumination 
    • 1.      cone of light that illuminates the specimen is seen as a bright background against which the image of the specimen must be contrasted
    • a.      good for specimens of high contrast (like stained sections), but it may not provide optimal visibility for other specimens
  19. Preparation of Specimens for Bright-field Light Microscopy                                                              
    specimens to be observed with LM divided into two: __
    whole mounts and sections
  20. Whole mount
    1.      intact object, either living or dead, and can consist of an entire microscopic organisms such as a protozoan or a small part of a larger organism
  21. Section
    very thin slice
  22. First step in preparing sections for Bright-field Light Microscopy
    a.      to prepare, cells are first killed by immersing the tissue in a chemical solution, called a fixative, which, if good, rapidly penetrates the cell membrane and immobilizes all of its macromolecular material so the structure of the cell resembles its living state
  23. Second step in preparing sections for Bright-field Light Microscopy
    a.      then, they are dehydrated by transfer through alcohols and embedded in paraffin (wax that is readily dissolved by organic solvents), which gives mechanical support during sectioning
  24. Third step in preparing sections for Bright-field Light Microscopy
    a.      Slides with paraffin sections are immersed in tuolene, which dissolves the wax, leaving a thin slice of tissue on the slide for staining, treatment, or etc. 
  25. Fourth step in preparing sections for Bright-field Light Microscopy
    a.      coverslip placed over tissue using mounting medium with same refractive index as the glass slide and coverslip