The light which is not absorbed is re-emitted in all directions, producing what is called scattering of the light; we mentioned the two extreme cases of total reflection and total transmission. Most cases are intermediate. If electron oscillations are equally possible in all directions, the incident unpolarized light beam will emerge essentially as it entered the suspension—except for the phase shift already mentioned. If, however, the particles or molecules in suspension are not isotropic, i.e., the electrons can oscillate more readily in one direction than in another, an incident unpolarized light beam will be split, because waves oscillating in one direction will have their phases shifted more than those oscillating in another direction. The net result is that so-called anisotropic molecules produce two plane polarized emergent beams. Since these emerge in somewhat different directions, the phenomenon is called double refraction or birefringence.

Clearly, a measurement showing birefringence tells us at once that the material being studied is anisotropic, and since the optical anisotropy is due to anisotropy in the permissible movements of electrons, we can infer something about the structure of the material.

The chief use of this phenomenon in biology presently is in the inverse fashion. We shine plane polarized light on the substance of unknown structure and see what happens to the plane of polarization. Further, if we line up the molecules of a substance (as can be done in several ways), then the refraction (bending of the light) measured along and at right angles to the direction of alignment will tell us even more about the electronic structure of the individual molecules. 

Birefringent structure may arise in several ways, of which a few will now be briefly presented.

Intrinsic birefringence

The chemical nature and structure of individual molecules may contribute birefringence if there is anisotropy in the movability of the electrons of the molecules. Chemical bonds themselves may be highly anisotropic, the C—C bond being a good example.

This bond, being a covalent bond due to shared electrons lying primarily between the atoms involved, is difficult to distort at right angles to the bond. On the other hand, the triple bond C = C has the atoms bound about three times as strongly, so that the distortion of electron positions which can be effected is mainly at right angles to the bond direction.

Form (or organizational) birefringence

As a result of grouping molecules in a regular array (or in a composite structure with an inherent regularity, e.g. nucleic acids) there may well occur directions of easier and harder electron movability in the structure as a whole.

Flow birefringence

If a solution is caused to flow, the viscous forces will tend to orient particles with their long axes in the direction of the flow. Spherical particles may even be distorted by these forces.

Thus, during the flow, there will be organizational birefringence whose magnitude will depend on the speed of the flow.

Consider the two separate cases of a collection of discs and a collection of rods, as sketched in the figure.

In the case of the discs, electrons can be moved more readily along the planes of the discs than out of the plane; therefore polarized light incident at right angles to the lineup axis will be affected more than polarized light incident along the axis. The case of the rods is precisely opposite, since electrons can more readily be moved along the lineup axis. Thus, the measurement of the birefringence for lined-up molecules tells us whether the molecules have a rodlike or a disclike shape and how they are oriented with respect to the lineup axis.

There are several important instances of the use of birefringence which permitted significant deductions about structures in biology. The case of muscle is the first one, in which the measurement of the birefringence permitted the conclusion that there are both isotropic and anisotropic regions in striated muscle—the bands called J and A are actually named for these properties. In the anisotropic region, the result of the birefringence measurement led to the conclusion that the actomyosin fibers must be oriented along the muscle fiber axis.

The second case is that of chloroplasts, whose birefringence showed them to be composed of a stack of discs long before the electron microscope made the structure visible.

The third case is that of DNA. By slowly drying a solution of DNA stretched over a hole by capillary forces, a fiber can be produced. The fiber birefringence shows that the individual elements of the DNA are lined up in the disc configuration, even though it was known from viscosity studies that the individual DNA molecules must be long and thin.

Thus it was possible to conclude that the elements composing the DNA—the nucleotides—must be arranged as flat discs perpendicular to the long axis of the molecule.