Light microscopy

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This article examine the microscope.

Resolution

.[1]
Where:

  • NA = 0.25 - 1.4, >1.0 is oil immersion.
  • R = resolving distance; smaller better.
  • gamma = wave length of light.

Closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger. R = 1.22 * {gamma \over ( D/2*f )}

Notes:

  • Larger 'D' is better.
  • Larger NA = better

f-number (N)

N = f/D.

Where:

  • N = f-number.
  • f = focal length.
  • D = diameter of entrance pupil.

At infinity: N = 1/(2*NA_i) f/D = 1/(2*NA_i) or 2*NA_i=D/f

N ---> smaller number = larger opening

Numerical aperature

http://en.wikipedia.org/wiki/Numerical_aperture NA = numerical aperature

NA = n*sin(theta)

Where:

  • n = index of refraction, n = 1.0 for air.
  • theta = half-angle of the max. cone of light

Most lens - 'achromats' -- only correct green. 'apochromatic' lenses - correct all colours.

Condenser

  • Condenser -- large flattened lens beneath the specimen
    • Iris diaphragm
      • Condenser diaphragm --> incr. contrast for resolution ---- large dia. good resol. bad contrast?
        • Field aperature diaphragm --> optical illumination.

Kohler illumination

Rationale

  • Maximize resolution. (???)

Procedure

  1. Any specimen on stage.
  2. Focus.
  3. Adj. field aperature (bottom) - to obscure periphery of field of view (FOV).
  4. Raise or lower condenser until field aperature diaphragm clearly focused
  5. +/-Center 'field aperature diaphragm - using condenser centering screws

References