by Roy Winsby

I thought a note on Refractive Index (often stated as R.I.) would not be out of place here for the newcomer to microscopy who might not understand the meaning of the term, which is a measurement of the deflection of light leaving one medium, say air, and entering another medium of a different density, say water, at an angle.

The simple example usually given in books is that of a stick put into clear water, the stick appearing to have entered the water at a different angle to the part still held in the air. It is the light illuminating the stick which has been bent and this always happens to light when it enters or leaves a different medium at an oblique angle.

The textbook explanation is that the R.I. figure is the measurement of the bending of light in a particular substance as against that in a vacuum which is designated as unity (= 1). All substances are denser than a vacuum, so all R.Is are greater than 1.

Lens manufacturers have to take account of R.Is in selecting glass for their lenses. We microscopists have to take account as regards the mounting medium for different types of specimens because the greater the difference between the respective R.Is of the specimen and the mountant used to affix it to the slide, the greater will be the contrast and thus the better the image when that slide is examined under the microscope.

The simplest example of this is that cover glasses have a R.I. of around 1.518 and Canada Balsam in Xylene a R.I. of around 1.524. These are quite close and if you mount some pieces of broken cover glass in Balsam and observe it under the microscope you will hardly see the broken bits of cover glass, because the light is not refracted or bent. If, however, the broken bits of cover glass were mounted in a medium of low refractive index such as Glycerine Jelly, R.I. 1.44, then they would be clearly visible.

Conversely diatoms, have a R.I. of 1.434, and would not be visible mounted in Glycerine Jelly and are best seen under a high refractive index mountant such as Hyrax, R.I. 1.71 to 1.82. Northern Biological Supplies Ltd and the stockists of their supplies sell two high resolution mountants for mounting diatoms, Dirax (new; I do not know the R.I. but I guess it will be around the same as Hyrax) which does not contain an inflammable solvent, and Naphrax, solvent toluene, R.I. at least 1.74.

Editors note: The Micscape Editor thanks Roy Winsby for allowing this article to be reproduced on the Web. Also thanks to Mike Samworth for preparing the Web version of the article.


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