Light microscopy
This article examine the microscope.
Resolution
.[1]
Where:
- R = resolving distance; smaller better.
- NA = 0.25 - 1.4, >1.0 is oil immersion.
- gamma = wave length of light.
Closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.
Notes:
- Larger 'D' is better.
- Larger NA = better
f-number (N)
N = f/D.
Where:
- N = f-number.
- f = focal length.
- D = diameter of entrance pupil.
At infinity: N = 1/(2*NA_i) f/D = 1/(2*NA_i) or 2*NA_i=D/f
N ---> smaller number = larger opening
Numerical aperature
http://en.wikipedia.org/wiki/Numerical_aperture NA = numerical aperature
NA = n*sin(theta)
Where:
- n = index of refraction, n = 1.0 for air.
- theta = half-angle of the max. cone of light
Most lens - 'achromats' -- only correct green. 'apochromatic' lenses - correct all colours.
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.
- Adj. field aperature (bottom) - to obscure periphery of field of view (FOV).
- Raise or lower condenser until field aperature diaphragm clearly focused
- +/-Center 'field aperature diaphragm - using condenser centering screws
References
- ↑ "Principles of Microscopy". http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html. Retrieved 21 January 2011.