Difference between revisions of "Light microscopy"

Latest revision as of 20:38, 23 April 2016

File:Optical microscope nikon alphaphot +.jpg

A light microscope.

This article examines light microscopy, abbreviated LM.

Resolution

$R=1.22*{\gamma \over {NA_{obj}+NA_{cond}}}$ .^[1]
Where:

$R$ = resolving distance; smaller better.
$NA_{obj}$ = numerical aperture of the objective; typically 0.25 - 1.4, >1.0 is oil immersion, it is usu. inscribed on the lens itself.
$NA_{cond}$ = numerical aperture of the condenser.
$\gamma$ = 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]

Stated differently:^[2]^[3]

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]
$NA=n*sin(theta)$ .

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:
$N=1/(2*NA_{i})$ .
$f/D=1/(2*NA_{i})$ .
$2*NA_{i}=D/f$ .

Numerical aperture

If one substitutes the above into the equation at the top:
$R=1.22*{\gamma \over (D/2*f)}$ .

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.

Depth of field

Abbreviated DOF.

DOF depends on the aperature (small is better).

Relation to other parameters:^[3]

Inverse relationship with resolution and brightness.
Related to contrast.
High magnification --> smaller depth of field.

Formula

$DOF={\lambda _{o}n \over NA^{2}}+{n \over M\cdot NA}e$ .^[5]

Where:

$\lambda _{o}$ = 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:

Hugin (sourceforge.net) - does focus stacking and stitching.

Image:

Example of focus stacking (WC).

Köhler illumination

Rationale

Even light distribution (avoid vignetting).

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]

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.

External links

Depth of field calculator (microscopyu.com).

[pom-1] 1.0 ^1.1 "Principles of Microscopy". http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html. Retrieved 21 January 2011.

[2] URL: http://www.microbehunter.com/2008/12/18/the-condenser-aperture-diaphragm/. Accessed on: 21 January 2011.

[grayfield_dof-3] 3.0 ^3.1 URL: http://www.grayfieldoptical.com/depth_of_fieldfocus.html. Accessed on: 27 May 2011.

[4] URL: http://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.

[5] URL: http://www.microscopyu.com/articles/formulas/formulasfielddepth.html. Accessed on: 27 May 2011.

[6] 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.

[1]

[2]

[3]

[4]

[5]

[6]

@@ 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==
-<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>
 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_{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>
-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.
 *Closing the condenser --> increases DOF & contrast -- but -- decreases resolution & brightness.
@@ Line 42: / Line 43: @@
 ====Numerical aperture====
 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:
@@ Line 58: / Line 59: @@
 ****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===
-*Maximize resolution. (???)
+*Even light distribution (avoid [[vignetting]]).
 ===Procedure===
@@ Line 68: / Line 99: @@
 #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).<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==
 *[[Basics]].
 *[[Microphotography]].
+*[[Electron microscopy]].
+*[[Polarization of light]].
 ==References==
 {{Reflist}}
+==External links==
+*[http://www.microscopyu.com/tutorials/java/depthoffield/index.html Depth of field calculator (microscopyu.com)].
 [[Category:Basics]]

Difference between revisions of "Light microscopy"

Latest revision as of 20:38, 23 April 2016

Contents

Resolution

Numerical aperture

NA and f-number

Numerical aperture

Lenses

Condenser

Depth of field

Formula

Increasing the DOF

Köhler illumination

Rationale

Procedure

Resolution

See also

References

External links

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