Light microscopy - Revision history

Michael: /* See also */

2016-04-23T20:38:59Z

Michael: /* Kohler illumination */

2014-10-17T05:12:20Z

Kohler illumination

← Older revision		Revision as of 05:12, 17 October 2014
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	*[http://commons.wikimedia.org/wiki/File:Focus_stacking_Tachinid_fly.jpg Example of focus stacking (WC)].		*[http://commons.wikimedia.org/wiki/File:Focus_stacking_Tachinid_fly.jpg Example of focus stacking (WC)].

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

	===Procedure===		===Procedure===

Michael at 05:07, 17 October 2014

2014-10-17T05:07:49Z

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	[[Image:Optical microscope nikon alphaphot +.jpg\|thumb\|~~350px~~\|A light microscope.]]		[[Image:Optical microscope nikon alphaphot +.jpg\|thumb\|250px\|A light microscope.]]
	This article examines '''light microscopy''', abbreviated '''LM'''.		This article examines '''light microscopy''', abbreviated '''LM'''.

Michael at 05:07, 17 October 2014

2014-10-17T05:07:14Z

← Older revision		Revision as of 05:07, 17 October 2014
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			[[Image:Optical microscope nikon alphaphot +.jpg\|thumb\|350px\|A light microscope.]]
	This article examines '''light microscopy''', abbreviated '''LM'''.		This article examines '''light microscopy''', abbreviated '''LM'''.

Michael: +abbrev.

2012-05-30T13:23:24Z

+abbrev.

← Older revision		Revision as of 13:23, 30 May 2012
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	This article examines '''light microscopy'''.		This article examines '''light microscopy''', abbreviated '''LM'''.

	==Resolution==		==Resolution==

Michael: /* Depth of field */ more

2011-05-30T15:49:26Z

Depth of field: more

← Older revision		Revision as of 15:49, 30 May 2011
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	*M = magnification.		*M = magnification.
	*e = resolution.		*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)].

	==Kohler illumination==		==Kohler illumination==

Michael: /* Depth of field */ more

2011-05-27T16:03:57Z

Depth of field: more

← Older revision		Revision as of 16:03, 27 May 2011
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	==Depth of field==		==Depth of field==
	*Abbreviated ''DOF''.		*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.

			*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>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>		</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.

	==Kohler illumination==		==Kohler illumination==

Michael: /* References */ +EL

2011-05-27T15:55:36Z

References: +EL

← Older revision		Revision as of 15:55, 27 May 2011
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	==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: +DOF section

2011-05-27T15:53:10Z

+DOF section

← Older revision		Revision as of 15:53, 27 May 2011
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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==

Michael: /* Resolution */ another ref.

2011-05-27T14:39:59Z

Resolution: another ref.

← Older revision		Revision as of 14:39, 27 May 2011
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	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>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.

← Older revision		Revision as of 20:38, 23 April 2016
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	*[[Microphotography]].		*[[Microphotography]].
	*[[Electron microscopy]].		*[[Electron microscopy]].
			*[[Polarization of light]].

	==References==		==References==