POLARIZED LIGHT MICROSCOPY
One of the most powerful tools forensic scientists have at their disposal is the polarizing light microscope, a tool of nearly infinite uses and applications. Sadly, in this age of computerized instrumentation, few scientists routinely use a polarized light microscope, or PLM. Something can be learnt about almost every kind of sample, from asbestos to zircon, by using PLM. The PLM exploits optical properties of mate rials to discover details about the structure and composition of materials, and these lead to its identification and characterization. Materials fall into one of two categories. The first are materials that demonstrate the same optical properties in all directions, such as gases, liquids, and certain glasses and crystals. These are isotropic materials. Because they are optically the same in all directions, they have only one RI. Light, therefore, passes through them at the same speed with no directional restrictions. The second category is anisotropic materials, which have optical properties that vary with the orientation of the incoming light and the optical structure of the mate rial. About 90% of all solid materials are anisotropic. The RIs vary in anisotropic materials depending both on the direction of the incident light and on the optical structure. Think of anisotropic materials as having a “grain,” like wood, with preferential orientations, as illustrated in Figure 4.10.
FIGURE 4.10 Isotropic materials have the same optical properties in all directions, whereas anisotropic ones have differing properties based on the incident light and the internal structure of the material. Anisotropic materials can be envisioned as having a “grain.
Because of their inhomogeneous internal structure, anisotropic materials divide light rays into two parts. PLM uses this to cause the light rays to interact in a way that yields information about the material. Light is emitted from a source in all directions; in the wave model of light, all directions of vibration are equally possible. If the light passes through a special filter, called a polarizer, then the only light that passes is that which vibrates in that “preferred” direction; light that vibrates in only one direction is called polarized light (see Figure 4.11). Human eyes are “blind” to the vibrational direction of light; it can be seen only by a color effect or by intensity. This may sound complicated, but chances are good that most people have seen polarized light—through polarized sunglasses! They reduce the glare, like off of a car hood on a sunny day, by filtering out all the light except for that which is traveling in the direction preferred by the orientation of the treated sunglass lens. All light that reflects off a flat surface is at least partially polarized. The easiest way to visualize polarization is to imagine a wave vibrating perpendicular to the direction in which it’s traveling. The light can move in two directions or vectors (the x and y components). In this simple example, assume the two components have exactly the same frequency (occurrence over time). The x and y components can differ in two other ways. The two components may differ in amplitude, and the two components may not have the same phase (they may not hit their peaks and troughs at the same time). When the shape is traced as the light wave, the light’s polarization state can be described as illustrated in Figure 4.11. A PLM uses two polarizing filters (or polarizers, sometimes called “polars,” for short), one called the “polarizer” (that’s obvious, isn’t it?) and the “analyzer” (for reasons that will become obvious). The polarizer sits beneath the stage and has its preferred vibration direction set left-to-right (sometimes called the “east–west”). The analyzer, aligned opposite to that of the polarizer (i.e., north–south), is located above the objectives; the analyzer can be manually slid into or out of the light path. If the analyzer is inserted with its orientation opposite to that of the polarizer (at right angles), what should be seen? Nothing. The filters are said to be crossed, and no light can pass through the microscope to the viewer’s eyes. The field of view appears black or very, very dark, as shown in Figure 4.12. Information can be obtained both in plane-polarized light (only the polarizer in place) or with crossed polarizers (polar izer and analyzer in place). Anisotropic materials split light into component light rays. Birefringence is the result of this division of light into at least two rays (the ordinary ray and the extraordinary ray) when it passes through certain types of material, depending on the polarization of the light. Two different refractive indices are assigned to the material for different polarization orientations (rotating the sample under the polarizing filter). Birefringence is quantified by
Δn=ne−no
where no is the RI for the ordinary ray and ne is the RI for the extraordinary ray. The difference in velocity of the ordinary and extraordinary rays is called retar dation and increases linearly with both the thickness of a specimen and with the birefringence. The greater the thickness, the greater the retardation (the thicker the specimen, the farther one ray lags behind the other) and the greater the difference between the refractive indices (that is, the higher the birefringence) to begin with, the greater the retardation. This can be related in an equation
r =t(n2 −n1)
where r is retardation, t is thickness, and n2 − n1 is birefringence.
FIGURE 4.11 Because of the orientation of the polarizing filter, only light rays that are in line with its orientation can pass through. This is how polarized sunglasses work, by filtering out scattered light rays and allowing only certain ones through. When the preferred orientations of the filters, sometimes called “polars,” are at right angles to each other, no light can pass through. Varying degrees of rotation will allow progressively more light through until the polars are aligned.
FIGURE 4.12 When an anisotropic material is placed under crossed polarizers and rotated on the optical axis of the microscope, polarization colors result. (left) A grain of sillimanite, a mineral component found in a soil sample from a crime scene (Academic Press, by permission). (middle, right) A section of brass metal with a fracture in polarized light and under cross polarizers (Carl Zeiss, with permission).
FIGURE 4.13 The Michel-Levy Chart devised in 1888 by a French geologist, August Michel-Levy.
When these out-of-phase waves of light strike the analyzer, it diffracts them into various colors depending on the wavelengths being added and subtracted through interference; they are called “interference colors.” These colors are caused by the interference of the two rays of light split by the anisotropic material interfering destructively with each other; that is, they cancel each other out to a greater or lesser degree. The colors produced are indicative of the specimen’s molecular organization. The birefringence of a specimen can be determined with the polarizing microscope by examining it between crossed polars. The characteristic birefringence of a given substance is the numerical difference between the maximum and minimum refractive indices. Birefringence will be greatest when the specimen’s molecular structure is aligned along its longitudinal axis and will be zero if they are randomly organized. A chart of diameter, birefringence, and retardation, pictured in Figure 4.13, is called a Michel-Levy Chart, after its inventor, Auguste Michel-Lévy (1844–1911). Michel-Levy, a French geologist, was born in Paris and became inspector-general of mines and director of the Geological Survey of France. He was distinguished for his research into the microscopic structure and origin of eruptive minerals; importantly, Michel-Levy was a pioneer in the use of the polarizing microscope for the determination of minerals. The chart assists in the identification of birefringent materials. One of the ingenious things about the chart is that if two of the parameters are known, the third can be calculated (using the equation listed previously). For more information about the Michel Levy Chart, see Delly (2003). For more information on microscopy, see “On the Web: Microscopy.
