Polarized Light Microscopy

What is polarized light microscopy?

Polarized light microscopy is a contrast-enhancing technique that dramatically improves the quality of an image acquired with birefringent materials when compared to other techniques such as brightfield and darkfield illumination, phase contrast, differential interference contrast, fluorescence, and Hoffman modulation contrast. Polarized light microscopes possess a high degree of sensitivity and can be used for both qualitative and quantitative studies targeted at a wide range of anisotropic specimens.

Open

How does polarized light microscopy work?

Polarized light microscopy (Figure 1(c)) is conducted by viewing the specimen between crossed polarizing elements inserted into the optical train before the condenser and after the objective. Assemblies within the cell having birefringent properties, such as the plant cell wall, starch granules, and the mitotic spindle, as well as muscle tissue, rotate the plane of light polarization, appearing bright on a dark background. As mentioned above, differential interference contrast operates by placing a matched pair of opposing Nomarski prisms between crossed polarizers, so that any microscope equipped for DIC observation can also be employed to examine specimens in plane-polarized light simply by removing the prisms from the optical pathway.

Open

There are two polarizing elements in a polarizing microscope (see Figure 2). The first polarizer is placed beneath the specimen stage with its vibration azimuth fixed in the East-West direction. Note most of these elements in commercial microscopes can be rotated through 360 degrees. Another polarized (the analyzer), which is usually aligned with a vibration direction oriented North-South (but again rotatable on some microscopes), is placed above the objective and can be moved into and out of the light path as needed. When both the polarizer and analyzer are inserted into the optical path, their vibration azimuths are positioned at right angles to each other. In this configuration, the polarizer and analyzer are crossed, with no light passing through the optical system and a dark background present in the eyepieces (Figure 2(a)). Linearly polarized light waves are generated by polarizers that filter out a privileged plane from the statistical confusion of vibrational directions prevailing in natural light. The two orthogonal components of light (ordinary and extraordinary waves) travel at different speeds through the specimen and, as a result, observe different refractive indices, a phenomenon known as birefringence (derived from the terms double or bi refraction). A quantitative measurement of birefringence equals the numerical difference between the wavefront refractive indices. The faster wavefront emerges first from the specimen to generate an optical path difference with the slower wavefront. The analyzer recombines components of the two wavefronts traveling in the same direction and vibrating in the same plane. This polarizing element ensures that the two wavefronts have the same amplitude at the time of recombination for maximum contrast.

The appropriate arrangement for polarized light imaging is relatively easy to implement on virtually any microscope. The polarizer beneath the condenser (near the aperture diaphragm) ensures that the specimen is illuminated with linearly polarized wavefronts that pass through the condenser (refer to Figure 2(a)). The analyzer (a second polarizer), which is oriented with the azimuth arranged at an angle of 90 degrees to that of the polarizer, is located behind the objective. The tube lens forms the intermediate image, which is projected through the eyepieces or onto a camera imaging plane. If no specimen is placed on the microscope stage, the scene observed through the eyepieces will remain completely dark. When illuminated, many specimens turn the vibration direction of the polarized light out of the plane produced by the polarizer. Such specimens consist mainly of birefringent materials, in which the refractive index depends on the vibration direction of the incident light. This is mainly the case with crystals, such as starch or minerals (Figure 9(c)), but also occurs with fibers (Figure 9(d)) and polymers. If such materials are viewed under the polarization microscope between the crossed polarizer and the analyzer, bright areas can be seen in the image because light is partially transmitted by the analyzer.

The polarized light microscope optical train can also include an auxiliary element known as a lambda or retardation plate (Figure 2(a) and 2(b)) that is used in quantitative analysis. In polarized light, this lambda plate converts contrast into colors. As in phase contrast, optical path differences give rise to colors, although this time with polarized light and birefringent material in the specimen. The path differences generated leads to an extinction of certain wavelengths in the light through interference (only certain colors remain from the white light and create beautiful, colored pictures).

References:

http://zeiss-campus.magnet.fsu.edu/articles/basics/contrast.html