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Oblique Illumination Dispersion Staining

Oblique Illumination Dispersion Staining

These are the colors seen when using oblique 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 Oblique Brightfield 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. Oblique 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 well over a large range of particle sizes, from a few micrometers to over 100 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:

Begin by establishing Koehler Illumination and screen off 1/2 to 2/3rds of the beam of light traveling from the condenser into the objective with the sub-stage condenser iris open fully. The particle will be seen with super resolution normal to the oblique light path and slightly reduced resolution parallel to the oblique path. If the microscope has a turret type phase contrast condenser this can be done by partially rotating the turret from the "0" position. The dispersion colors will be evident on oposite sides of the field of view.

Characteristic Features:

One advantage of this technique is that no accessories to a standard microscope are required for its use though a phase contrast turret condenser can simplify its use. 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.