Difference between revisions of "Light microscopy"

Jump to navigation Jump to search

Light microscopy (view source)

Revision as of 20:38, 23 April 2016

2,763 bytes added ,  20:38, 23 April 2016
→‎See also
 
(16 intermediate revisions by the same user not shown)
Line 1: Line 1:
This article examine the '''microscope'''.
[[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:
*<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><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.
*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 aperture.


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.
Line 31: Line 39:
<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==
Line 54: Line 59:
****Field aperature diaphragm --> optical illumination.
****Field aperature diaphragm --> optical illumination.


==Kohler 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===
*Maximize resolution. (???)
*Even light distribution (avoid [[vignetting]]).


===Procedure===
===Procedure===
#Any specimen on stage.
#Any specimen on stage.
#Focus.
#Focus.
#Adj. field aperature (bottom) - to obscure periphery of field of view (FOV).
#Adjust field aperture (bottom) - to obscure periphery of field of view (FOV).
#Raise or lower condenser until field aperature diaphragm clearly focused.
#Raise or lower condenser until field aperture diaphragm clearly focused.
#+/-Center 'field aperature 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]]
Michael
48,479

edits

Retrieved from "https://librepathology.org/wiki/Special:MobileDiff/3773...43871"

Navigation menu