# User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/01/Brief Summary of Some Concepts in Microscopy

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• Quick summery of some concepts in optics

## Köhler Illumination

Köhler Illumination [1] is the proper alignment of incident or illuminating light, this technique yields optimal illumination for entire field of view.

Process

• Focus on image
• Close the field diaphragm so that the edges of the diaphragm are in the field of view
• Use condenser focus to bring the edges of the diaphragm into the best focus
• Open the field diaphragm so that the edges are just beyond the field of view
• Adjust condenser diaphragm to introduce the proper amount of contrast into the sample. Careful, too much contract can introduce artifacts into the image
• Adjust light intensity by using neutral density filters rather than changing intensity of light source. Changing the power of the light source will alter the spectrum while neutral density filters block light at all wavelengths.

## DIC Microscopy

Differential interference contrast (DIC)[2] microscopy is a technique designed to enhance contrast in a sample. This technique can gain information about the optical density of the sample and can produce an image that gives the appearance of a three-dimensional physical relief. The final image appears three dimensional with light and dark shadows. This method is very useful as it allows for observing the the entire shape of the object. The picture on the side shows the light path of a reflected DIC microscope (taken from [3])

The light path of a reflected DIC microscope.

Process:

• Unpolarized light enters microscope, passes through a 45° polarizer, and is thus 45° polarized.
• Polarized light enters a Nomarski-modified Wollaston prism. The prism separates light into two rays polarized 90 degrees from each other, so now there are two beams polarized at 0° and 90°.
• The two rays are focused by the condenser for passage through the sample. These two rays are focused so they will pass through two adjacent points in the sample, about 200 nm apart. The sample is now illuminated by two coherent light sources (with 0° and 90° polarized) that are slightly misaligned.
• The two rays travel through different and adjacent ares of the sample. The path lengths of the two rays differ where index of refraction and/or thickness of the sample differ. This change in path length causes a change in phase relative to the other ray.
• The rays are passed through the objective lens and then focused on the Nomarski-modified Wollaston prism, where the rays are recombined and now polarized at 135°. The two rays have passed through different points on the sample, so interference occurs between rays of light that went through adjacent points. The recombination of the light causes "optical differentiation". When combined the phase difference between the two rays results in interference.

## Numerical Aperture

The numerical aperture[4] of an optic tells the maximum diameter of a beam for which it is functional. It is defined by

$\mathrm{NA} = n \sin \theta\;$

where n is the index of refraction of the medium in which the optic is placed (air, oil, etc) and θ is the half-angle of the maximum cone of light that can enter/exit the optic.