The mounting medium can have a strong impact on how the specimen appears under the microscope.
If you want to observe a specimen under the microscope, then you have to add a mounting medium. The mounting medium is important for several reasons:
- Stability: It mechanically stabilizes and supports the specimen. Delicate structures can not break off.
- Preservation: It prevents moisture and bacteria from reaching the specimen, which might decompose it.
- Improves image quality: The refractive index (RI) of the mounting medium can have a significant impact on the image quality.
It is this last point that I want to talk about more in this article. We first have to understand, that the specimen itself also has a certain refractive index. The differences between the refractive index of the specimen and the surrounding mounting medium now influences how the specimen appears under the microscope.
If you do not use a mounting medium, then the specimen is surrounded by air. These specimens are dry-mounted. There is a large difference in refractive index. This results in strong refraction of light where the specimen and the mounting medium meet. As a consequence, some parts of the specimen appears darker and this can cover up some of the details and color of the specimen. One of the most extreme cases occurs, when the specimen is dry mounted (without mounting medium, only air). In this case there is a large difference between the refractive index between the specimen and the air. As a consequence, dark fringes appear around the specimen, where the specimen and the air meet.
The image above illustrates this case. The pollen grains are much darker on the edges. Air bubbles in water are another example. While both air and water are transparent, the air bubbles are quite well visible because of the dark ring around the bubble. In short, a large difference in RI causes artifacts as light is scattered and can not be picked up by the objective. In most cases, this scattering is considered bad as they cover up important details. On the other hand, it does increase contrast for difficult to see specimens.
Small differences in refractive index between specimen and mounting medium will result in reduced scattering of light and the natural colors of the specimen appear stronger. The specimen also will appear more transparent and brighter. This is the case when mounting the specimen in water.
For most cases, this is the preferred situation, but there are situations where this can be seen as a disadvantage. Specimens that generally lack color and are small will be more difficult to see (in brightfield). In these cases the refraction would be helpful in increasing the contrast. Bacteria are such a case. The are small, transparent and lack color. They become visible because the RI difference and the resulting refraction make them appear darker (even in brightfield). If there is absolutely no difference in Refractive Index, then the specimen is only visible in bright field if it has color.
Euparal as a Mounting Medium
Euparal is a commonly used to mount insects, but it can also work as a general-purpose mounting medium. Drying times are long, up to 6 weeks. Euparal can tolerate small amounts of moisture in the specimen, and specimens can be transferred directly from alcohol into Euparal.
Pollen mounted in Euparal started to shrink and therefore appear smaller in size. Kinks and folds also became visible. These artifacts are produced because the (non-water based) Euparal has withdrawn moisture from the pollen.
Clear Nail Polish as a Mounting Medium
Clear nail polish showed a similar, but more pronounced effect as Euparal. The deformations of the pollen are very clearly visible. Evidently the solvent of the nail polish also removed significant amounts of water from the specimen. The nail polish itself lost some of its volume during drying and started to shrink as well. Air bubbles also became visible in the nail polish. The pollen has darker edges and this means that the refractive index difference is larger than with Euparal.
Phase contrast vs. Bright field
The choice of mounting medium can also be important when working with a phase contrast microscope.
When you have a bright field microscope then the Refractive Index difference between specimen and mounting medium should be small. The color contrast should be large, however. This allows you to open the conducer diaphragm to increase resolution.
Phase contrast microscopes depend on a difference of Refractive Index between the specimen and the surrounding medium. The microscope converts differences in Refractive Index into brightness differences. Ideal phase contrast specimens have a Refractive Index difference but no color. In this case the specimen color does not interfere with the brightness difference created by the phase contrast optics.
What can we learn from these observations?
- First, permanently mounting a specimen is not only important for slide storage. The mounting medium significantly influences the transparency, resolution and shape of the specimen.
- Second, the choice of the mounting medium depends on the type of specimen to be observed and on the type of microscopic technique to be used. For phase-contrast work the refractive index of the mounting medium should be different from the refractive index of the specimen. For bright-field work the refractive indexes should be similar. Large differences in refractive index can lead to the dark fringes as seen in the air-mounted specimens.