Dispersion staining is a group of analytical techniques based on the analytical interpretation of color effects resulting from differences in the rate of change of the refractive index with wavelength (dispersion) between a standard material and an unknown. It is an optical effect and does not involve a chemical reaction or require a chemical affinity as do chemical staining techniques. Dispersion staining has been widely used in the study of everything from dental caries to hazardous materials identification.

Significant advances in the technique of free particle analysis have occurred since the last major study of dispersion staining by G. B. Hoidale. That study, conducted in 1964, provided a description of dispersion staining as it was understood at that time and included an extensive list of references. In the intervening years dispersion staining has been much more widely applied as an analytical technique. There is now a better appreciation of the material properties underlying the dispersion of the refractive index and there have been many improvements in the detection and characterization of these properties.

The dispersion of a material is related to the nature of the bond energies between ions in the molecules of that material and the energy of the wavelength of light used to measure those properties. The dispersion of a material is typically plotted on a graph with the abcissa being the refractive index and the ordinate being the wavelength scaled as a function of the reciprocal of the wavelength squared (see Figure 1). Bond energies between three and five electron volts result in a very steep dispersion curve. Materials with higher bond energies have dispersion curves with a more horizontal slope, as the bond energies increase the slope of the dispersion curve decreases.

There are five basic methods of dispersion staining. These are Becke’ line dispersion staining, oblique illumination dispersion staining, condenser stop dispersion staining, objective stop dispersion staining, and phase contrast dispersion staining.

Becke’ line dispersion staining was first documented in 1911 by F. E. Wright. He referred to the colored Becke’ lines that could be seen around a particle when the refractive index of the particle and that of the surrounding liquid in which it was mounted matched for some visible wavelengths but not for others. Figures 2 and 3 illustrate the path of a parallel bundle of rays (substage condenser iris closed down) through a particle mounted in a liquid of lower and of higher refractive index respectively. Figure 4 shows the appearance of the Becke’ line as the plane of focus is moved above that of the edge of the particle by defocusing the microscope. The Becke’ Line is the bright band of light that moves into the particle if the particle has the higher refractive index or out into the surrounding liquid if the liquid has the higher refractive index. If the refractive index of the particle (nobj) and the liquid (nsur) match at one wavelength (λ0) but the liquid has a steeper dispersion curve than the solid (generally the case) then two Becke’ lines are created (see Figure 5). The Becke’ line moving into the particle is the combination of all of the wavelengths for which the particle has the higher refractive index. These are all the wavelengths toward the red end of the spectrum from the matching wavelength. The color of this Becke’ line will vary from pale yellow if λ0 is in the deep blue to dark red if λ 0 is in the orange-red part of the spectrum. The Becke’ line that moves into the surrounding liquid is the combination of all the wavelengths for which the liquid has the higher refractive index. These are all the wavelengths toward the blue end of the spectrum from the matching wavelength. The color of this Becke’ Line will vary from deep blue if λ 0 is in the blue part of the spectrum to pale blue if λ0 is in the orange-red part of the spectrum.

Wright also proposed the oblique illumination method. The colors created are the same but they color significant areas of the opposite sides of the particle when the particle is in sharp focus (see Figure 6). By using a narrow range of angles for the oblique illumination this technique can be very effective and has the advantage of keeping the particle sharply in view and of creating a larger colored area which is easier to interpret.

Another dispersion staining method was introduced by G. C. Crossmon during the 1940’s. It involved the use of a darkfield stop in the substage condenser of the microscope. A further improvement of this method was made during the 1970’s by using a more limited cone of illumination (R. G. Speight). This was accomplished by using a phase annulus (see Figure 7) rather than the simpler darkfield stop. The particle appears on a black background in the complimentary color to that of the matching wavelength. The color ranges from pale yellow, to gold, to shades of magenta, and finally to blue as λ0 moves from deep blue (400 nanometers) through dark red (700 nanometers).

In the 1950’s Yu. A. Cherkasov in the Soviet Union devised another technique. He began by noting the distribution of the rays that gave rise to the colored Becke’ lines as they passed through the back focal plane of the objective (see Figure 8). By placing opaque screens of different configurations at the back focal plane he could control which set of rays could pass to create the final image of the object (see Figure 9). This technique was further developed by K. M. Brown and Walter McCrone and is the most common dispersion staining technique used today, though often not the most appropriate. The color sequence when a central stop is used is the same as that for the condenser stop technique above but this technique is more sensitive to smaller particles and generates brighter colors. One disadvantage is the greater loss of resolving power, the shape of object is more difficult to see. When the oblique stop is used the color sequence is the same as for oblique illumination with the same advantages and disadvantages noted for the central stop. The annular stop creates a particle image in the wavelength for which the liquid and the particle match in refractive index in theory. In fact the matching refractive index is mixed with the direct ray and a green color dominates the series of colors that is difficult to characterize as to the location of the matching wavelength. The central stop is almost exclusively used when objective stop dispersion staining is applied.

In the late 1950’s and early 1960’s K. G. Schmidt in Germany wrote a paper on the color effects seen when using phase contrast microscopy (see Figure 10). These effects were due to the combination of dispersion and the phase shift in the rays of light passing through the particle compared to those passing through the mounting medium. His papers noted the effects but did not explain the origin of the colors. As a result his observations were not generally repeatable. The dependence of the phase shift as a function of particle thickness (see Figure 11) was the principle parameter missing in Schmitt’s work. For particles less than ten micrometers in thickness a reliable and repeatable sequence of colors is created that can be used to determine the wavelength at which a particle and its surrounding mounting liquid match. The sequence of colors is basically the same as for the colored Becke’ Lines except the blue colors become the color of the entire particle and the red colors become a halo around the particle. This system has the advantage of showing the color effect without any loss of resolving power in the image. This technique is a significant addition to dispersion staining test procedures and is vastly superior to the objective stop method for the characterization of small particles or fibers as in the analysis of asbestos.

The literature on dispersion staining methods is woefully deficient in detailed descriptions of the mechanisms involved. Cherkosov’s paper on objective stop methods and a paper by Speight on condenser stop methods are two notable exceptions. Dispersion staining methods are being widely applied, not just for bulk asbestos analysis but for other types of analysis also (E. R. Crutcher).

REFERENCES

• 1. Hoidale, G. B., U.S. ARMY MICROFICHE, AD 603 019, 1964.
• 2. Wright, F. E., Publication No. 158, Carnegie Inst., Washington, D. C., pp. 87-, 1911.
• 3. Crossmon, G. C. “Microscopical Distribution of Corundum Among its Natural and Artificial Associates”, ANALYTICAL CHEMISTRY, Vol. 20, No. 10, 1948.
• 4. Speight, Richard G., “An Alternative Dispersion Staining Technique”, THE MICROSCOPE, Vol. 25, 1977.
• 5. Cherkasov, Yu. A., “Application of ‘Focal Screening’ to Measurements of Indices of Refraction by the Immersion Method”, INTERNATIONAL GEOLOGICAL REVIEW, Vol.2,1960.
• 6. Brown, K. M. and W. C. McCrone, “Dispersion Staining”, THE MICROSCOPE, Vol. 13 and 14, 1963.
• 7. Schmidt, K. 0., “Phase Contrast Microscopy and Dispersion Staining”,STAUB, Vol. 18, 1958.
• 8. Crutcher, E. R., “The Role of Light Microscopy in Aerospace Analytical Laboratories”, PROCEEDINGS OF THE NINTH SPACE SIMULATION SYMPOSIUM, 1977.
• 9. Crutcher, E. R., “Optical Microscopy: An Important Tool for Particulate Receptor Source Apportionment”, PROCEEDINGS OF THE AIR POLLUTION CONTROL ASSOCIATION, 1981.

Dispersion Staining Color by the Value of λ0 The top bar shows the color that corresponds to the λ0 match for central stop and Darkfield dispersion staining. The two lower bars show the two colors that are seen with colored Becke Line, Oblique, and Phase Contrast dispersion staining for a corresponding λ0. The colors will vary a bit depending on the specific configuration of the microscope and the relative shape of the dispersion curves. Fig.1: Dispersion curves for high density liquids and optical glass standards Fig.2: Light path through particle, refractive index of particle greater than liquid Fig.3: Light path through particle, refractive index of particle less than liquid Fig.4: Becke' lines Fig.5: Becke' lines dispersion staining Fig.6: Oblique illumination dispersion staining Fig.7: Condenser stop dispersion staining Fig.8: Wavelength distribution about λ0 caused by dispersion, SSC iris closed Crutcher（1989）による『Introduction to dispersion staining microscopy』から

Figure 9: Objective stop dispersion staining Crutcher（1989）による『Introduction to dispersion staining microscopy』から

Figure 10: Phase contrast dispersion staining Figure 11: Phase shift caused by particle in surrounding medium Crutcher（1989）による『Introduction to dispersion staining microscopy』から