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Darkfield Dispersion Staining

Glass Sphere in Matching Refractive Index Liquid When Viewed with Darkfield Illumination

When darkfield illumination is used the wavelengths for which the particle has the higher refractive index and the wavelengths for which the liquid has the higher refractive index combine to form the color at the edge of the particle. This is the color seen when using darkfield illumination and the refractive index of the particle and the refractive index liquid match at a wavelength of 589 nanometers, the Sodium D-Line.

Transmitted Darkfield Illumination


Dispersion Staining is an optical staining technique created by differences in the dispersion of the refractive indices for a particle and the liquid in which it is mounted. Darkfield dispersion staining is one of the five methods of dispersion staining. It results in a single color, similar to the objective central stop dispersion staining method, to assess the wavelength at which the particle and liquid match. It produces brilliant colors and is very effective for particles larger than about 10 micrometers with most standard illumination sources.

Conditional Requirments:

This approach works best with a mounting medium that has a steep dispersion curve. Most liquids with refractive indices above 1.60 meet that requirement. There are "high dispersion" liquids sold commercially designed specifically for dispersion staining. These sets normally start at a refractive index of 1.500 and go up to about 1.700. The particles of interest are mounted in one of these liquids that matches the refractive index of the particles at some visible wavelength. High dispersion liquids can also be made by mixing cinnamic aldehyde (R.I. about 1.62) with triethyl phosphate (R.I. 1.406), or methylene Iodide (R.I. 1.737). A less expensive set of high dispersion liquids can be made with cinnamon oil, also called cassia oil (R.I. about 1.60) and clove oil (R.I. about 1.53) or caster oil (R.I. about 1.48). These oils can generally be purchased at any local drugstore. When liquids are mixed it is good to test them against standard glasses or minerals on a regular basis. The commercial refractive index liquids are designed for long term stability.

The particles must be mounted under a coverslip to optimize the effects and minimize in interference cause by any optical anomaly in an unmounted specimen.

Microscope Configuration:

The easiest way to configure the microscope for darkfield illumination is to purchase a set of plug-in stops from the microscope manufacturer or purchase their phase contrast condenser. Begin by establishing Koehler Illumination with the condenser stop out. Insert the stop for the objective being used and check the back focal plane of the objective by using a phase telescope, the Bertrand lens, a pinhole eyepiece, or by removing the eyepiece and viewing down the tube. The condenser stop should block all direct light from entering the objective. The stop may need to be centered for this to be the case. When orthoscopic viewing is resumed the background should be black. If it is not then the stop is too small or it is not centered properly. The colors are clearly visible with the particle in sharp focus.

Characteristic Features:

One advantage of this technique is that very simple and relatively inexpensive accessories are required to configure a standard microscope. The stop can be easily made out of any opaque material suspended in the plane of the sub-stage condenser iris and centered so that it meets the criteria stated above. Most manufacturers make the required stops designed for their specific microscopes. A second advantage is that the particle is seen in full, even super resolution for the objective being used and the particle is in sharp focus at the time the colors are seen. A disadvantage is that the colors are best for particles larger than about 10 micrometers. Another disadvantage is that the particles should be isolated, not stacked or in too complex a field of view.