Difference between revisions of "Light microscopy"

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


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>
*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===
===Numerical aperture===
<math>R = 1.22 * {gamma \over ( D/2*f )}</math>.  
NA = numerical aperature.


Notes:
General formula for NA:<ref>URL: [http://en.wikipedia.org/wiki/Numerical_aperture http://en.wikipedia.org/wiki/Numerical_aperture]. Accessed on: 21 January 2011.</ref><br>
*Larger 'D' is better.
<math>NA = n*sin(theta)</math>.
*Larger NA = better.


f-number (N)
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.
N = f/D.


Where:
Where:
*N = f-number.
*N = f-number, e.g. f 1.2, f 1.4, f 11.
**Smaller N = larger opening.
*f = focal length.
*f = focal length.
*D = diameter of entrance pupil.
*D = diameter of entrance pupil.
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<math>N = 1/(2*NA_i)</math>.<br>
<math>N = 1/(2*NA_i)</math>.<br>
<math>f/D = 1/(2*NA_i)</math>.<br>
<math>f/D = 1/(2*NA_i)</math>.<br>
<math>f/D = 2*NA_i=D/f</math>.
<math>2*NA_i = D/f</math>.


N ---> smaller number = larger opening.
====Numerical aperture====
If one substitutes the above into the equation at the top:<br>
<math>R = 1.22 * {gamma \over ( D/2*f )}</math>.


==Numerical aperature==
Notes:
NA = numerical aperature.<ref>URL: [http://en.wikipedia.org/wiki/Numerical_aperture http://en.wikipedia.org/wiki/Numerical_aperture]. Accessed on: 21 January 2011.</ref>
*Larger 'D' is better.
 
*Larger NA = better.
<math>NA = n*sin(theta)</math>.
 
Where:
*n = index of refraction, n = 1.0 for air.
*theta = half-angle of the max. cone of light


==Lenses==
==Lenses==

Revision as of 22:19, 21 January 2011

This article examine the microscope.

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]

  • 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 aperature.

General formula for NA:[3]
.

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. 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.

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://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.
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