Difference between revisions of "Light microscopy"

Revision as of 21:57, 21 January 2011

This article examine the microscope.

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 aperature of the condenser.
$gamma$ = wave length of light.

Closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.^[1]

Numerical aperture

$R=1.22*{gamma \over (D/2*f)}$ .

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

N ---> smaller number = larger opening.

Numerical aperature

NA = numerical aperature.^[2]

$NA=n*sin(theta)$ .

Where:

n = index of refraction, n = 1.0 for air.
theta = half-angle of the max. cone of light

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.

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

↑ ^1.0 ^1.1 "Principles of Microscopy". http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html. Retrieved 21 January 2011.
↑ URL: http://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.

[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://en.wikipedia.org/wiki/Numerical_aperture. Accessed on: 21 January 2011.

[1]

[2]

@@ Line 2: / Line 2: @@
 ==Resolution==
-<math>R = 1.22 * {gamma \over {NA_{obj} + NA_{cond}}}</math>.<ref>{{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:
-*R = resolving distance; smaller better.
+*<math>R</math> = resolving distance; smaller better.
-*NA = 0.25 - 1.4, >1.0 is oil immersion.
+*<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.
-*gamma = wave length of light.
+*<math>NA_{cond}</math> = numerical aperature of the condenser.
+*<math>gamma</math> = wave length of light.
-Closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.<br>
+Closure of the condenser diaphragm results in a loss of resolution, i.e. R is larger.<ref name=pom/><br>
+===Numerical aperture===
 <math>R = 1.22 * {gamma \over ( D/2*f )}</math>.
@@ Line 25: / Line 28: @@
 *D = diameter of entrance pupil.
-At infinity:
+At infinity:<br>
-N = 1/(2*NA_i).
+<math>N = 1/(2*NA_i)</math>.<br>
-f/D = 1/(2*NA_i)    or    2*NA_i=D/f.
+<math>f/D = 1/(2*NA_i)</math>.<br>
+<math>f/D = 2*NA_i=D/f</math>.
 N ---> smaller number = larger opening.