Microscope Activities, 20: Refractive Index Determination

In the past, Hooke College of Applied Sciences offered a microscopy workshop for middle school and high school science teachers. We thought that these basic microscope techniques would be of interest not only for science teachers, but also for homeschoolers and amateur microscopists. The activities were originally designed for a Boreal/Motic monocular microscope, but the Discussion and Task sections are transferable to most microscopes. You may complete these 36 activities in consecutive order as presented in the original classroom workshop, or skip around to those you find interesting or helpful. We hope you will find these online microscope activities valuable.

EXPERIMENT 20: Refractive Index Determination


To learn how to determine the relative refractive index/indices of any sample



Materials Needed

Polarizer; Orange filter; Selection of various liquids.
Optional: Set of refractive index liquids.


Put your homemade Polarizer (Experiment 15) in the slot at the top of the light exit port of your Boreal/Motic microscope.

Using your 10X objective, focus on any specimen; our specimen is quartz. Now swing over to your 40X objective, and close down the aperture diaphragm; focus slightly above or below best focus until you see a halo of light around the outside edge of a quartz particle.

Insert an orange filter in the microscope light path. This can be a photographic filter; any orange light will be adequate to start with. Professional microscopists use a 589 nm precision line interference filter for this, but they are expensive (you can buy them from Edmund Scientific). For introductory work, an inexpensive acetate filter, such as the Roscolux #23 Orange mounted between glass in a 35 mm slide mount will be more than adequate. Figure 20-1 illustrates such an orange filter being placed over the light exit port; this should be right over the polarizer, which itself is in the groove of the light exit port. The cap analyzer is not used; this lighting condition using only the polarizer is called plane polarized light.

sodium d 589 nm filter, Becke line
Figure 20-1.

Now, looking at the edge of the particle being illuminated with light vibrating east/west, and composed of light wavelengths in the orange region of the spectrum, focus UP (that is, from best focus to a higher plane than the particle; to do this, the stage will be moved downward slightly). In which direction does the halo move—into the quartz or out of the quartz, into the medium? Record the result.

Repeat this exercise using calcite as a sample; rotate the slide and notice what happens to the contrast of the individual particles as you rotate the sample. It will almost disappear at one point in the rotation, and be very contrasty at another point 90º away. Stop at the most contrasty view. Repeat the test of focusing at the edge, and then focusing UP, noting which way the halo moves—into the particle, or into the medium?


The halo produced on closing the aperture diaphragm is called the Becke Line, named after the Austrian mineralogist. This Becke line always moves into the higher refractive index on focusing UP. In the case of quartz or glass, we know that the refractive index of the mounting medium is nD25°C =1.662; you always have to know the optical properties of any mounting medium that you use. While looking at quartz, and focusing UP, the Becke line was seen to move out of the particle, and into or toward the medium. The Becke line always moves toward whichever has the higher refractive index. It moved toward the medium, therefore the mounting medium has the higher refractive index. However, it is the particle that we are interested in, so we reason: if the medium has the higher index then the particle has a lower index. The symbol for refractive index in “n”, so the way this fact about quartz is expressed is: nD25°C <1.662.

The contrast is an indicator of how far away the true refractive index is; if a particle and its mounting medium have exactly the same refractive index, the particle will “disappear”; it will not be seen at all, and there can be no contrast. The farther apart the refractive index of a particle and its medium are, the more contrast between the two. Thus, the Becke line can be used to quickly determine the relative refractive index of the sample, compared to the known refractive index of the medium.

In analytical microscopy and particle identification work, a set of about ninety or more bottles of refractive index liquid, varying from about nD25°C =1.300 to nD25°C =1.700 or more, with intervals of 0.002, is available so that an unknown sample may be successively mounted in different liquids until one is found in which the sample “disappears”; then, knowing the refractive index of the liquid from the bottle label, the refractive index of the sample is known.

Most materials in the world have more than one characteristic refractive index—they may have two or three. That is why plane polarized light produced by our polarizer is essential. The refractive index of the sample being measured is that one which is parallel to the polarizer vibration direction. In the case of calcite, you noticed when you rotated it, its appearance alternated between disappearing and being very contrasty. In the contrasty orientation you observed the Becke line to move out of the particle on focusing UP. How do we interpret this?

There must be more than one refractive index (in fact, there are two in this sample); one of them must be very near to nD25°C =1.662, because at some point the calcite disappeared; the other one must be much lower than nD25°C =1.662, because the Becke line moved into the medium, and the contrast was very high. The two characteristic refractive indices of calcite are nD25°C =1.658 (see how close to nD25°C =1.662 this is when it disappeared; that direction in the particle was parallel to the polarizer vibration direction) and nD25°C =1.486 (compared to nD25°C =1.662 of the medium, this is much lower; that’s why the high contrast).

It is all just a bit more involved, because the temperature and wavelength of light affect the refractive index, and the values found for the refractive index need to be considered in connection with the crystal morphology and orientation to be analytically useful; nevertheless, being able to determine the relative refractive index, as you have done in this experiment, is the first big step in identifying any unknown.


Configure your Boreal/Motic as described in the Procedure section, and determine the relative refractive indices of quartz and calcite, as directed. Now try this with the synthetic polyester textile fiber dacron©; for this, and all fiber samples, orient the fiber first E/W (i.e., parallel to the polarizer vibration direction) to obtain the relative refractive index for the length of the fiber; and then rotate it N/S to obtain the relative refractive index across the width of the fiber; the two refractive indices, n parallel and n perpendicular characterize the fiber; the numerical difference between the two refractive indices is known as the birefringence, and is a characteristic identification value.


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