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Difference between revisions of "Light microscopy"
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+DOF section
(→Resolution: another ref.) |
(+DOF section) |
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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== | ||