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Positive Phase Contrast Dispersion Staining

Positive Phase Contrast Dispersion Staining

These are the colors seen when using phase contrast illumination with the 1.66 optical glass standard in Cargille Melt Mount 1.662 refractive index liquid. The wavelength at which this glass and this liquid match is 670 nanometers. The difference in dispersion is 0.012.

Transmitted Positive Phase Contrast Illumination, 20X Objective


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. Phase Contrast dispersion staining is one of the five methods of dispersion staining. It is used as a screening technique and for detailed dispersion staining analysis. It works best for particles that are smaller in their shortest diameter than 10 micrometers.

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:

This method requires a phase contrast objective and a matching phase contrast condenser stop. If only one objective is to be purchased it is best to use the 20X positive phase objective. If a turret phase condenser is purchased it will facilitate phase contrast, darkfield, and oblique dispersion staining methods.

Characteristic Features:

One advantage of this method is that the particle is in sharp focus and in high resolution when the colors are evident. Another advantage is that there are two colors, The sum of the wavelengths for which the particle has the higher refractive index and the sum of the wavelengths for which the liquid has the higher refractive index. This helps to bracket the matching wavelength. A further advantage is that the depth of field is extremely shallow. Single small particles can be optically isolated and their colors seen easily. This is a unique property among the five dispersion staining methods. Its disadvantage is the limit on the particle size, it works best for smaller particles or fibers. This is ideal for use in the characterization of asbestos in construction materials. Asbestos fibers are small and often intimately mixed with other materials. Phase contrast dispersion staining can isolate stacked particles and particles in complex mixtures, showing the dispersion colors associated with that single small particle, which is sharply in focus and clearly visible.