Dispersion staining
The optical properties of all liquid and solid materials change as a function of the wavelength of light used to measure them. This change as a function of wavelength is called the dispersion of the optical properties. The graph created by plotting the optical property of interest by the wavelength at which it is measured is called a dispersion curve.
The dispersion staining is an analytical technique used in light microscopy that takes advantage of the differences in the dispersion curve of the refractive index of an unknown material relative to a standard material with a known dispersion curve to identify or characterize that unknown material. These differences become manifest as a color when the two dispersion curves intersect for some visible wavelength. This is an optical staining technique and requires no stains or dyes to produce the color. Its primary use today is in the confirmation of the presence of asbestos in construction materials but it has many other applications.
Types
There are five basic optical configurations of the microscope used for dispersion staining. Each configuration has its advantages and disadvantages. The first two of these, Becke` line dispersion staining and oblique dispersion staining, were first reported in the United States by F. E. Wright in 1911 based on work done by O. Maschke in Germany during the 1870s. The five dispersion staining configurations are:All of these configurations have the same requirements for the preparation of the sample to be examined. First, the substance of interest must be in intimate contact with the known reference material. In other words, the clean solid must be mounted in a reference liquid, one mineral phase must be in intimate contact with the reference mineral phase, or the homogenous liquid must contain the reference solid. Most applications involve a solid mounted in a reference liquid. Second, dispersion colors will only be present if the two materials have the same refractive index for some wavelength in the visible spectrum and they have very different dispersions curves for the refractive index. Finally, the sample must be properly mounted under a coverslip to minimize any other optical effect that could complicate the interpretation of the color seen. Once these criteria are met the sample is ready to be examined.
The starting configuration of the microscope for all of these methods is properly adjusted Köhler illumination. Some additional adjustments are required for each of the methods.
Becke' line dispersion staining
The Becke' Line method takes advantage of the fact that particles behave basically like lenses because they tend to be thinner at the edges than they are at the center. If the particle has a higher refractive index than the liquid surrounding it then it behaves as a convex lens and focuses a parallel beam of light on the side opposite the source of the light. Looking through the microscope this is seen as a bright ring of light, the Becke` Line, moving in from the edge as the particle is dropped out of focus by increasing the distance between the stage of the microscope and the objective. If the stage is moved closer to the objective then the particle behaves like a magnifying glass and the image of the Becke` Line is magnified and it appears outside the particle.Image:Dispersion Staining Becke line.jpg|thumb|right|These are the colored Becke` lines of a glass sphere that matches the refractive index of the mounting medium at a wavelength of 589 nanometers.
A requirement for this method is that the incoming beam of light is as parallel as possible. This requires the closing down of the sub-stage condenser iris. Closing the sub-stage condenser iris decreases the resolution of the particle and increases the depth of field over which other objects may interfere with the effect seen. For large particles this is not a significant limitation but for small particles it is a problem.
When the conditions for dispersion staining are met then the particle has a high refractive index in the red part of the spectrum and a lower refractive index in the blue. This is because liquids tend to have a steeper dispersion curve than colorless solids. As a result, when the particle is dropped out of focus the red wavelengths are focused inward. For the blue wavelengths the particle behaves like a concave lens and the blue Becke` Line moves out into the liquid.
The color of these two bands of light will vary depending on where the particle and liquid match in refractive index, the location of λo. If the match is near the blue end of the spectrum then the Becke' Line moving into the particle will contain nearly all of the visible wavelengths except blue and will appear as a pale yellow. The Becke` Line moving out will appear a very dark blue. If the match is near the red end of the spectrum then the Becke` Line moving into the particle will appear dark red and the Becke` Line moving out will appear pale blue. If the λo is near the middle of the visible wavelengths then the Becke` Line moving into the particle will be orange and the Becke` Line moving out will be sky blue. The colors seen can be used to very precisely determine the refractive index of the unknown or confirm the identity of the unknown, as in the case of asbestos identification. Examples of this type of dispersion staining and the colors shown for different λo's can be seen at . The presence of two colors helps to bracket the wavelength at which the refractive index matches for the two materials.
Image:Dispersion Staining Color Pairs2.JPG|thumb|Chart 1: These are the dispersion staining colors associated with different matching wavelengths when using any of the methods that generate a color pair.
The Becke' Line method of dispersion staining is primarily used as an exploratory technique. As a particle field is scanned and the fine focus is constantly adjusted and flash of color around or in a particle is noted and one of the other methods can be used to sharpen the sensitivity in determining the matching wavelength. For large particles the colored Becke` Lines may be sufficiently distinct to determine the lo with the required accuracy. For very large particles this may be the best method because it is least sensitive to other types of optical interferences.
Oblique illumination dispersion staining
Oblique illumination dispersion staining is the result of refraction and the convex shape of most particles. With oblique illumination the beam of light illuminating the sample is directed at an oblique angle through the sample. This enhances the resolution of structural details in the particle that are oriented at right angles to the incident beam of light while sacrificing some of the resolution of features parallel to the direction of the beam. Because of this orientation of the beam the relative refractive index of the particle and the mounting liquid becomes apparent. The wavelengths for which the liquid has the higher refractive index are refracted into the front lens of the objective from the side of the particle nearest the side from which the light is coming. If the particle has a higher refractive index for all visible wavelengths then this side of the particle is dark. The side farthest from the source of the light shows all the wavelengths for which the particle has the higher refractive index. These effects are seen with the particle in sharp focus. This is a significant advantage over the Becke` line method because the particle doesn't have to be defocus to see the colors and generally the colors are more distinct than are the Becke` line dispersion colors. The colors seen with this type of dispersion staining are about the same as those with the Becke` Line method shown in Chart 1. Examples of this type of dispersion staining and the colors shown for different λo's can be seen at . The presence of two colors helps to bracket the wavelength at which the refractive index matches for the two materials.Darkfield illumination dispersion staining
Darkfield illumination dispersion staining is the result of the image of the particle being formed only by the light that is refracted while all the direct light impinging on the specimen is oriented at such an angle that it misses the front lens of the objective. Image:Dispersion Staining Darkfield.jpg|thumb|right|This is the color shown by a glass sphere that matches the refractive index of the mounting medium at a wavelength of 589 nanometers when using darkfield dispersion staining.The result is that the background is black. All of the features of objects in the field of view that don't match the refractive index of the mounting medium appear as bright white. When a particle is mounted in a liquid that matches its refractive index somewhere in the visible wavelengths then those wavelengths are not refracted by the particle and are not collected by the objective. The image of the object is formed by all of the wavelengths that remain. These wavelengths combine to produce a single color that can be used to indicate which band of wavelengths are missing. Examples of this type of dispersion staining and the colors shown for different λo's can be seen at the . This method is more difficult to interpret due to the single color rather than two bracketing colors but is relatively accurate near the center of the visible range.
Image:Dispersion Staining Color Pairs.JPG|thumb|Chart 2: These are the dispersion staining colors associated with different λo's when using any of the methods that generate a single color.
Phase contrast dispersion staining
Phase contrast dispersion staining requires that a phase contrast objective with the appropriate phase annulus in the substage condenser be used to see the effect. It takes advantage of the fact that the rays of light that are not shifted in phase by the presence of the object are separated from the phase shifted rays at the back focal plane of the objective.Image:Dispersion Staining Phase Contrast.jpg|thumb|right|These are the colors shown by a glass sphere that matches the refractive index of the mounting medium at a wavelength of 589 nanometers when using phase contrast dispersion staining. These unaffected rays are then significantly decreased in intensity. With “Positive Phase Contrast”, the particle appears colored from the contributing wavelengths for which the mounting medium has the higher refractive index. Because of the physical size of the phase plate and its image onto the back focal plane of the objective where it is modified, a halo is formed around the particle. This halo takes on the color of the combined wavelengths for which the particle has the higher refractive index. The colors seen with this type of dispersion staining are about the same as those with the Becke` Line method shown in Chart 1. Examples of this type of dispersion staining and the colors shown for different λo's can be seen at the . The presence of two colors helps to bracket the wavelength at which the refractive index matches for the two materials.