Difference between revisions of "Light microscopy"
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==Resolution== | ==Resolution== | ||
<math>R = 1.22 * {gamma \over {NA_{obj} + NA_{cond}}}</math>.<ref name=pom>{{cite web |url=http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html |title=Principles of Microscopy |author= |date= |work= |publisher= |accessdate=21 January 2011}}</ref> | <math>R = 1.22 * {\gamma \over {NA_{obj} + NA_{cond}}}</math>.<ref name=pom>{{cite web |url=http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html |title=Principles of Microscopy |author= |date= |work= |publisher= |accessdate=21 January 2011}}</ref> | ||
<br> | <br> | ||
Where: | Where: | ||
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*<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 aperture 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. | ||
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><ref>URL: [http://www.grayfieldoptical.com/depth_of_fieldfocus.html http://www.grayfieldoptical.com/depth_of_fieldfocus.html]. Accessed on: 27 May 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 name=grayfield_dof>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. | ||
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====Numerical aperture==== | ====Numerical aperture==== | ||
If one substitutes the above into the equation at the top:<br> | If one substitutes the above into the equation at the top:<br> | ||
<math>R = 1.22 * {gamma \over ( D/2*f )}</math>. | <math>R = 1.22 * {\gamma \over ( D/2*f )}</math>. | ||
Notes: | Notes: | ||
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***Condenser diaphragm --> incr. contrast for resolution ---- large dia. good resol. bad contrast? | ***Condenser diaphragm --> incr. contrast for resolution ---- large dia. good resol. bad contrast? | ||
****Field aperature diaphragm --> optical illumination. | ****Field aperature diaphragm --> optical illumination. | ||
==Depth of field== | |||
*Abbreviated ''DOF''. | |||
*It depends on the aperature (small is better).<ref name=grayfield_dof>URL: [http://www.grayfieldoptical.com/depth_of_fieldfocus.html http://www.grayfieldoptical.com/depth_of_fieldfocus.html]. Accessed on: 27 May 2011.</ref> | |||
**Inverse relationship with resolution and brightness. | |||
**Related to contrast. | |||
<math>DOF = { \lambda_o n \over NA^2}+{ n \over M \cdot NA } e | |||
</math>.<ref>URL: [http://www.microscopyu.com/articles/formulas/formulasfielddepth.html http://www.microscopyu.com/articles/formulas/formulasfielddepth.html]. Accessed on: 27 May 2011.</ref> | |||
==Kohler illumination== | ==Kohler illumination== | ||
Revision as of 15:53, 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]
- 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.
- Condenser diaphragm --> incr. contrast for resolution ---- large dia. good resol. bad contrast?
- Iris diaphragm.
Depth of field
- Abbreviated DOF.
- It depends on the aperature (small is better).[3]
- Inverse relationship with resolution and brightness.
- Related to contrast.
.[5]
Kohler illumination
Rationale
- Maximize resolution. (???)
Procedure
- Any specimen on stage.
- Focus.
- Adjust field aperture (bottom) - to obscure periphery of field of view (FOV).
- Raise or lower condenser until field aperture diaphragm clearly focused.
- +/-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
- ↑ 1.0 1.1 "Principles of Microscopy". http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html. Retrieved 21 January 2011.
- ↑ URL: http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/. Accessed on: 21 January 2011.
- ↑ 3.0 3.1 URL: http://www.grayfieldoptical.com/depth_of_fieldfocus.html. Accessed on: 27 May 2011.
- ↑ URL: http://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.
- ↑ URL: http://www.microscopyu.com/articles/formulas/formulasfielddepth.html. Accessed on: 27 May 2011.
- ↑ 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.