Section 5.1.1.3.1. Crystals between crossed polarizers

Most protein crystals are birefringent and are brightly coloured in polarized light. In order to view these effects, crystals (in their mother liquor) are set on a microscope stage with a Nicol prism (polarizing material) between the light source and the microscope slide (the polarizer). Another Nicol prism is set between the crystal and the eyepiece (the analyser). The crystal should not be in a plastic container, since this would produce too many colours. If the vibration plane of the analyser is set perpendicular to that of the polarizer (to give `crossed Nicols'), no light will pass through in the absence of crystals, and the background will be dark. If the crystal is isotropic, the image will remain dark as the crossed Nicol prisms are rotated. If, however, the crystal is birefringent (with two refractive indices), the crystal will appear coloured except at four rotation positions (90° apart) of the crossed Nicol prisms, where the crystal and background will be dark (extinguished). At these positions, the vibration directions of the Nicol prisms coincide with those of the crystal. If one is looking exactly down a symmetry axis of a crystal that is centrosymmetric in projection (such as a tetragonal or hexagonal crystal), the crystal will not appear birefringent, but dark. By noting the external morphology of the crystal with respect to its angle of rotation, one can often deduce the directions of the unit-cell axes in the crystal (Hartshorne & Stuart, 1960). Examination of a crystal under crossed Nicol prisms can also provide information on crystal quality. For example, sometimes the components of a twinned crystal extinguish plane-polarized light independently. Other methods of examining crystals include Raman spectroscopy (Kudryavtsev et al., 1998).