An idea for high-RI diatom mountant
Moderators: rjlittlefield, ChrisR, Chris S., Pau
An idea for high-RI diatom mountant
I have been frustrated by the lack of availability and price of the more famous high-refractive-index diatom mountants. High RI is needed because diatoms have a refractive index around 1.35-1.4, not that different from water, so they don't show up well. The famous mountants like Naphrax have RI around 1.73-1.74.
By accident today I found that people who work with gems use a high-RI liquid with their refractometers to reveal the RI of their gemstones, a useful clue to a gemstone's identity. This liquid has an RI of 1.8 and is readily available on eBay. Does anyone have any experience with it in microscopy?
By accident today I found that people who work with gems use a high-RI liquid with their refractometers to reveal the RI of their gemstones, a useful clue to a gemstone's identity. This liquid has an RI of 1.8 and is readily available on eBay. Does anyone have any experience with it in microscopy?
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Re: An idea for high-RI diatom mountant
It's kind of dark in color, smells bad and a bit toxic, shouldn't be put on skin. I think it may also be corrosive--it certainly can break down the lead glass of a hemicylinder if left on it.
Re: An idea for high-RI diatom mountant
Hmm, a liquid that breaks down glass may not be the ideal mountant! The dark color probably won't matter though, since it will be very thin.
Re: An idea for high-RI diatom mountant
Reading more about the hemicylinders, it seems this is an especially soft and reactive glass. Maybe regular glass would not corrode?
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Re: An idea for high-RI diatom mountant
Lead glass is definitely more delicate than your average glass, so it is something to keep in mind.
- MarkSturtevant
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Re: An idea for high-RI diatom mountant
No idea, but my first thought would be to try polarized light to enhance contrast.
Mark Sturtevant
Dept. of Still Waters
Dept. of Still Waters
Re: An idea for high-RI diatom mountant
Will try that, Mark, as well as oblique lighting.
Re: An idea for high-RI diatom mountant
Diatoms frustule are made of amorphous silica, so in principle polarized light would do nothing with them. Pol requires birefringence.MarkSturtevant wrote: ↑Wed Jan 26, 2022 12:48 pmNo idea, but my first thought would be to try polarized light to enhance contrast.
Other contrast techniques able to enhance small differences in light path and/or speed will work, mainly DIC and DF but also oblique and likely Hoffman MC. Phase contrast also will work although at risk of halos.
Pau
Re: An idea for high-RI diatom mountant
I often photograph diatoms "mounted" in air, which gives more contrast in normal brightfield illumination than any high-RI mountant. I don't do it for the contrast per-se, I just don't want the diatoms mounted (yet) as they're destined for storage on keeper slides first. But it's often handy to be able to move individual forms and get shots of the same specimen from different angles - especially when I've only found one example. I use an inverted scope for this, with the diatoms sat on the top surface of a large coverslip - lens looks through the glass from underneath.
The greater the RI difference between mountant and subject (diatom), the greater the contrast. The RI delta between silica and air is ~0.4, but ~0.3 between silica and the usual high-RI mountants.
No free lunch though. In air, the amount in focus (apparent depth of field) is less than in a high-RI mountant. Focus stacks of diatoms shot in air need significantly more frames to cover a given depth in the subject than when shot in a mountant.
Edit: PS. this technique would lend itself quite well to experimenting with diatoms sat in a small puddle of the jewellers stuff you mentioned. Though it would have to be cleaned off afterwards. Certainly an idea that's worth a tinker one day. I'll be back...
The greater the RI difference between mountant and subject (diatom), the greater the contrast. The RI delta between silica and air is ~0.4, but ~0.3 between silica and the usual high-RI mountants.
No free lunch though. In air, the amount in focus (apparent depth of field) is less than in a high-RI mountant. Focus stacks of diatoms shot in air need significantly more frames to cover a given depth in the subject than when shot in a mountant.
Edit: PS. this technique would lend itself quite well to experimenting with diatoms sat in a small puddle of the jewellers stuff you mentioned. Though it would have to be cleaned off afterwards. Certainly an idea that's worth a tinker one day. I'll be back...
Re: An idea for high-RI diatom mountant
I remember your impressive very trhee-dimensional-looking photo of a diatom in air (no coverslip) with your 100x. Shooting in ari is definitely something I want to try. The thing is that in order to get higher resolution, an immersion objective is a necesity. I have water, glycerine, and oil immersion objectives to try.....
Re: An idea for high-RI diatom mountant
Well, it's easy to *actually* mount diatoms in air. Stick a small shim washer on the slide (I use 13mm X 7mm X 0.1mm), mount your diatoms on a coverslip, then stick that on the washer with the diatoms inside. Use Debe's to stick the diatoms to the coverslip. It will also stick a simple strew, so no micromanipulators needed. You can immerse objective and condenser with that.
But I think this is moot. Won't the air/glass interfaces on the inside defeat what the immersion oils do outside? So low angle rays just bounce off that? Not sure...
But I think this is moot. Won't the air/glass interfaces on the inside defeat what the immersion oils do outside? So low angle rays just bounce off that? Not sure...
Re: An idea for high-RI diatom mountant
I would not use air in any part of the system for an immersion objective. I was just saying that mounting in air (at any stage) is not going to acheive maximum resolution.
Re: An idea for high-RI diatom mountant
Yeah, understood. I was questioning my own speculation, not your intention.
How about an ultra darkfield condenser? I used to have an NA1.2/1.4 Zeiss one, but lost it in a move. I've waited nearly ten years for a replacement to turn up - nothing yet. Anyway, they work great and by exposing for the highlights you can pull out a lot of contrast and detail where it counts (particularly around the smallest details). Invert the final image for a normal brightfield look.