Everyday Scientist

update on Nikon objective immersion oils

August 30, 2018 at 8:41 am | sam | everyday science, hardware, review

A few years ago, I compared different immersion oils. I concluded that Nikon A was the best for routine fluorescence because: (A) it had low autofluorescence, (B) it didn’t smell, (C) it was low viscosity, and (D) the small plastic dropper bottles allowed for easy and clean application.

Unfortunately, my two favorites, Nikon A and NF, were both discontinued. The oil Nikon replaced these with is called F. But I don’t love this oil for a few reasons. First, it’s fairly stinky. Not offensive, but I still don’t want my microscopes smelling if I can help it. Second, I’ve heard complaints from others that Nikon F can have microbubbles (or maybe crystals?) in the oil, making image quality worse. Finally, dried F oil hardens over time, and can form a lacquer unless it is cleaned off surfaces very well. That said, F does have very low fluorescence, so that’s a good thing.

I explored some alternatives. Cargille LDF has the same optical properties as Nikon F (index of refraction = 1.518 and Abbe Ve = 41). But LDF smells terrible. I refuse to have my microscope room smell like that! Cargille HF doesn’t smell and has similar optical properties, but HF is autofluorescent at 488 and 405 nm excitation, so it adds significant background and isn’t usable for sensitive imaging.

At the recommendation of Kari in the UCSF microscopy core (and Caroline Mrejen at Olympus), I tried Olympus Type F, which also has an index of refraction of 1.518 and an Abbe number of 40.8, which is compatible with Nikon. The Olympus oil had very low autofluorescence, on par with Nikon A, NF, and F. (I also tested low-fluorescence oils Leica F and Zeiss 518F, but their dispersion numbers are higher (Ve = 45-46), which can cause chromatic aberration and may interfere with Perfect Focus.)

I used to love the low viscosity of Nikon A (150 cSt), because it allowed faster settling after the stage moved and was less likely to cause Perfect Focus cycling due to mechanical coupling to thin or light samples, plus it was easier to apply and clean. Nikon NF was higher viscosity (800 cSt). Olympus F is higher than Nikon A (450 cSt), but acceptable.

Finally, Olympus F comes is an easy to use applicator bottle: instead of a glass rod that can drip down the side of the vial if you’re not careful, the Olympus F is in a plastic bottle with a dropper. It’s not quite as nice as the 8 cc dropper bottles that Nikon A used to come in, and I don’t love the capping mechanism on the Olympus F, but I’ll survive.

I plan to finish up our last bottle of Nikon A, then switch over to Olympus F. We also have a couple bottles of Nikon NF remaining, which I will save for 37C work (the higher viscosity is useful at higher temperatures).

UPDATE:

Some people claim that type A was simply renamed type N. I don’t think that’s true. First of all, I couldn’t get Perfect Focus on our Ti2 to work with Nikon type N oil. Second, the autofluorescence of Nikon type N (right) was way higher than Olympus type F (left) or the old Nikon type A, at least at 405 and 488 nm:

So I’ll stick with Olympus type F. :)

UPDATE 2:

Here are some example images. These are excited with 640 (red), 561 (red), 488 (green), and 405 nm (blue) and the display ranges are the same for each sample. (The dots are single fluorophores on the glass.) You can see that Cargille HF is slightly more autofluorescent (especially at 405 nm) than either the old Nikon A or Olympus type F. This matches what Cargille states for HF: “Slightly more fluorescent than Type LDF.”