Difference between revisions of "Light microscopy"

From Libre Pathology
Jump to navigation Jump to search
m (fix lead in)
(→‎Resolution: another ref.)
Line 12: Line 12:
It follows from the above equation that, closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.<ref name=pom/><br>
It follows from the above equation that, closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.<ref name=pom/><br>


Stated differently:<ref>URL: [http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/ http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/]. Accessed on: 21 January 2011.</ref>
Stated differently:<ref>URL: [http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/ http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/]. Accessed on: 21 January 2011.</ref><ref>URL: [http://www.grayfieldoptical.com/depth_of_fieldfocus.html http://www.grayfieldoptical.com/depth_of_fieldfocus.html]. Accessed on: 27 May 2011.</ref>
*Opening the condenser --> increases resolution & brightness -- but -- decreases depth of field (DOF) & contrast.
*Opening the condenser --> increases resolution & brightness -- but -- decreases depth of field (DOF) & contrast.
*Closing the condenser --> increases DOF & contrast -- but -- decreases resolution & brightness.
*Closing the condenser --> increases DOF & contrast -- but -- decreases resolution & brightness.

Revision as of 14:39, 27 May 2011

This article examines light microscopy.

Resolution

.[1]
Where:

  • = resolving distance; smaller better.
  • = numerical aperture of the objective; typically 0.25 - 1.4, >1.0 is oil immersion, it is usu. inscribed on the lens itself.
  • = numerical aperture of the condenser.
  • = 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]
.

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:
.
.
.

Numerical aperture

If one substitutes the above into the equation at the top:
.

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.

Kohler illumination

Rationale

  • Maximize resolution. (???)

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).[5]

See also

References

  1. ↑ 1.0 1.1 "Principles of Microscopy". http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html. Retrieved 21 January 2011.
  2. ↑ URL: http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/. Accessed on: 21 January 2011.
  3. ↑ URL: http://www.grayfieldoptical.com/depth_of_fieldfocus.html. Accessed on: 27 May 2011.
  4. ↑ URL: http://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.
  5. ↑ URL: http://www.biocompare.com/Articles/ApplicationNote/1668/Recent-Approaches-To-%E2%80%98Super-Resolution-Microscopy-Utilizing-Camera-Detection.html. Accessed on: 2 May 2011.
Retrieved from "https://librepathology.org/w/index.php?title=Light_microscopy&oldid=5628"