Something a bit different with this slide. On it are three strews, mounted using different mountants – [Canada] Balsam, Styrax, Hyrax. Species are given as Pleurosigma angulatum, Pleurosigma quadratum, Surirella gemma, Nitzschia sp. (and others). No makers name. These images are not mean to be the ultimate in resolving power, I kept the imaging conditions the same for each one, and also although they were denoised and sharpened I have not tweaked the contrast or brightness (more on that in a minute).
Olympus BHB microscope using 450nm LED light. 20x Nikon Plan Apo objective NA 0.65. Olympus Aplanat Achromat condenser, brightfield lighting. 2.5x Nikon CF PL photoeyepiece. Monochrome converted Nikon d850 camera. Three stacks prepared in Zerene (Pmax).
What’s going on here? The three mountants give different end results. Hyrax is very high contrast (higher than the other two). Styrax is lower contrast than Hyrax, but slightly more than Balsam. This is an effect based on the refractive index (RI) of the mountant and how it compares with the diatoms. As the RI difference between the mountant and diatom increases the visiblity (contrast) improves. There is a nice discussion on this in RB McLaughlin, Special Methods in Light Microscopy, 1977, p141. In this the RI of diatoms is given as 1.434. The RI’s and ‘visibility index’ of the mountants is shown below;
Balsam, RI 1.526, visibility index 0.092
Styrax, RI 1.580, visibility index 0.146
Hyrax, RI 1.700, visibility index 0.266
The higher the visibility index, the more visible the diatoms become. So, as we can see, as the RI of the mountant increases and becomes more different to the diatom, the visibility index improves. Yay, this is what we see, and reality matches theory.
However some thoughts on this. RI depends on wavelength of light being used. McLaughlin mentions that RI is typically reported for 589.3nm light (yellow). I’m imaging in blue at 450nm. Canada Balsam RI is higher at 450nm vs 589.3 nm (about 0.03 difference from what I have been able to find). So far I have not been able to find the RI of Styrax or Hyrax at 450nm, but again these are likely to be slightly higher than the values for 589.3nm. Despite this though, it is a useful comparison and shows how RI of the mountant can improve diatom contrast.