Light microscopy

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This article examines light microscopy, abbreviated LM.

Resolution

R = 1.22 * {\gamma \over {NA_{obj} + NA_{cond}}}.[1]
Where:

  • R = resolving distance; smaller better.
  • NA_{obj} = numerical aperture of the objective; typically 0.25 - 1.4, >1.0 is oil immersion, it is usu. inscribed on the lens itself.
  • NA_{cond} = numerical aperture of the condenser.
  • \gamma = wave length of light.

It follows from the above equation that, closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.[1]

Stated differently:[2][3]

  • Opening the condenser --> increases resolution & brightness -- but -- decreases depth of field (DOF) & contrast.
  • Closing the condenser --> increases DOF & contrast -- but -- decreases resolution & brightness.

Numerical aperture

NA = numerical aperture.

General formula for NA:[4]
NA = n*sin(theta).

Where:

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

NA and f-number

N = f/D.

Where:

  • N = f-number, e.g. f 1.2, f 1.4, f 11.
    • Smaller N = larger opening.
  • f = focal length.
  • D = diameter of entrance pupil.

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

Numerical aperture

If one substitutes the above into the equation at the top:
R = 1.22 * {\gamma \over ( D/2*f )}.

Notes:

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

Lenses

  • Most lens = 'achromats' -- only correct green.
  • 'Apochromatic' lenses - correct all colours; very expensive.

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.

Depth of field

  • Abbreviated DOF.
  • DOF depends on the aperature (small is better).

Relation to other parameters:[3]

  • Inverse relationship with resolution and brightness.
  • Related to contrast.
  • High magnification --> smaller depth of field.

Formula

DOF = { \lambda_o n \over NA^2}+{ n \over M \cdot NA } e
.[5]

Where:

  • \lambda_o = illuminating light wavelength.
  • n = refractive index of the medium, 1.0 for air.
  • NA = numerical aperature (objective).
  • M = magnification.
  • e = resolution.

Increasing the DOF

  • DOF can be increased by focus stacking.

Software:

Image:

Köhler illumination

Rationale

Procedure

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

Resolution

  • Usual light microscopes are limited to about 0.2 micrometres.
    • Coming is "Super-resolution microscopy" - using high speed CCDs (charge-coupled devices).[6]

See also

References

External links