Polarized Light Microscopy

Polarized Light Microscopy - Java Tutorial

Polarized light microscopy is a useful method to generate contrast in birefringent specimens and to determine qualitative and quantitative aspects of crystallographic axes present in various materials. The beautiful kaleidoscopic colors displayed by specimens under crossed polarizers arises as a result of the interference between light waves passing through the specimen.

This interactive tutorial simulates 360-degree rotation of birefringent samples through crossed polarizers in a polarizing microscope. To operate this tutorial, first select a sample from the Choose A Specimen pull-down menu. Next, use the Angle slider to rotate the sample stage through 360 degrees. The image will change depending on sample orientation, with the birefringent crystals changing from bright to total extinction and then back again. The blue arrow buttons allow movement of the sample stage in 10 degree increments.

There are two polarizing filters in a polarizing microscope - the polarizer and analyzer. The polarizer is situated below the specimen stage usually with its permitted vibration direction fixed in the left-to-right, East-West direction, although this is usually rotatable through 360 degrees. The analyzer, usually aligned North-South but again rotatable on some microscopes, is sited above the objectives and can be moved in and out of the light path as required. When both the analyzer and polarizer are in the optical path, their permitted vibration directions are positioned at right angles to each other. In this configuration, the polarizer and analyzer are said to be crossed, with no light passing through the system and a dark field of view present in the eyepieces.

Polarization colors result from the interference of the two components of light split by the anisotropic specimen and may be regarded as white light minus those colors that are interfering destructively. The two components of light travel at different speeds through the specimen and have different refractive indices, or refringences. Birefringence is the numerical difference between these refringences. The faster beam emerges first from the specimen with an optical path difference (OPD), which may be regarded as a "winning margin" over the slower one. The analyzer recombines only components of the two beams traveling in the same direction and vibrating in the same plane. The polarizer ensures that the two beams have the same amplitude at the time of recombination for maximum contrast.

There is constructive and destructive interference of light in the analyzer, depending on the OPD on the specimen and the wavelength of the light, which can be determined from the order of polarization color(s). This relies on the properties of the specimen, including the thickness difference between the refractive index and the birefringence of the two beams, which has a maximum value dependent on the specimen and on the direction of travel of light through the specimen. Optical path differences can be used to extract valuable "tilt" information from the specimen.

Superimposed on the polarization color information is an intensity component. As the specimen is rotated relative to the polarizers, the intensity of the polarization colors varies cyclically, from zero (extinction) up to a maximum after 45 degrees and back down to zero after a 90-degree rotation. That is why a rotating stage and centration are provided, which are critical on a polarizing microscope. Centration of the objective and stage ensures that the center of the stage rotation coincides with the center of the field of view, a great convenience, as anyone who has tried to manage without it will know.

Whenever the specimen is in extinction, the permitted vibration directions of light passing through are parallel with those of either the polarizer or analyzer. This can be related to geometrical features of the specimen, such as fiber length, film extrusion direction, and crystal faces. In crossed polarizers, isotropic materials can be easily distinguished from anisotropic materials as they remain permanently in extinction (remain dark) when the stage is rotated through 360 degrees.