Light microscopy

This article examine the microscope.

Resolution

$R=1.22*{gamma \over {NA_{obj}+NA_{cond}}}$.
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