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Difference between revisions of "Light microscopy"
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This article | [[Image:Optical microscope nikon alphaphot +.jpg|thumb|250px|A light microscope.]] | ||
This article examines '''light microscopy''', abbreviated '''LM'''. | |||
==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: | ||
| Line 8: | Line 9: | ||
*<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> | 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: | ||
| Line 58: | Line 59: | ||
****Field aperature diaphragm --> optical illumination. | ****Field aperature diaphragm --> optical illumination. | ||
== | ==Depth of field== | ||
*Abbreviated ''DOF''. | |||
*DOF depends on the aperature (small is better). | |||
Relation to other parameters:<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. | |||
*High magnification --> smaller depth of field. | |||
===Formula=== | |||
<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> | |||
Where: | |||
*<math>\lambda_o</math> = 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: | |||
*[http://hugin.sourceforge.net/ Hugin (sourceforge.net)] - does ''focus stacking'' and ''stitching''. | |||
Image: | |||
*[http://commons.wikimedia.org/wiki/File:Focus_stacking_Tachinid_fly.jpg Example of focus stacking (WC)]. | |||
==Köhler illumination== | |||
===Rationale=== | ===Rationale=== | ||
* | *Even light distribution (avoid [[vignetting]]). | ||
===Procedure=== | ===Procedure=== | ||
| Line 68: | Line 99: | ||
#Raise or lower condenser until field aperture diaphragm clearly focused. | #Raise or lower condenser until field aperture diaphragm clearly focused. | ||
#+/-Center 'field aperture diaphragm - using condenser centering screws. | #+/-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).<ref>URL: [http://www.biocompare.com/Articles/ApplicationNote/1668/Recent-Approaches-To-%E2%80%98Super-Resolution-Microscopy-Utilizing-Camera-Detection.html http://www.biocompare.com/Articles/ApplicationNote/1668/Recent-Approaches-To-%E2%80%98Super-Resolution-Microscopy-Utilizing-Camera-Detection.html]. Accessed on: 2 May 2011.</ref> | |||
==See also== | ==See also== | ||
*[[Basics]]. | *[[Basics]]. | ||
*[[Microphotography]]. | *[[Microphotography]]. | ||
*[[Electron microscopy]]. | |||
*[[Polarization of light]]. | |||
==References== | ==References== | ||
{{Reflist}} | {{Reflist}} | ||
==External links== | |||
*[http://www.microscopyu.com/tutorials/java/depthoffield/index.html Depth of field calculator (microscopyu.com)]. | |||
[[Category:Basics]] | [[Category:Basics]] | ||