Difference between revisions of "Light microscopy"
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===Numerical aperture=== | ===Numerical aperture=== | ||
NA = numerical | NA = numerical aperture. | ||
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> | 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> | ||
| Line 65: | Line 65: | ||
#Any specimen on stage. | #Any specimen on stage. | ||
#Focus. | #Focus. | ||
# | #Adjust field aperture (bottom) - to obscure periphery of field of view (FOV). | ||
#Raise or lower condenser until field | #Raise or lower condenser until field aperture diaphragm clearly focused. | ||
#+/-Center 'field | #+/-Center 'field aperture diaphragm - using condenser centering screws. | ||
==See also== | ==See also== | ||
Revision as of 22:20, 21 January 2011
This article examine the microscope.
Resolution
.[1]
Where:
- = resolving distance; smaller better.
- = numerical aperture of the objective; typically 0.25 - 1.4, >1.0 is oil immersion, it is usu. inscribed on the lens itself.
- = numerical aperture of the condenser.
- = 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]
- 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:[3]
.
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:
.
.
.
Numerical aperture
If one substitutes the above into the equation at the top:
.
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.
- Condenser diaphragm --> incr. contrast for resolution ---- large dia. good resol. bad contrast?
- Iris diaphragm.
Kohler illumination
Rationale
- Maximize resolution. (???)
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.
See also
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.
- ↑ URL: http://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.