Silicone immersion objectives - any experience?

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viktor j nilsson
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Silicone immersion objectives - any experience?

#1 Post by viktor j nilsson » Fri Jan 15, 2021 10:34 am

Hello,
Has anyone here had any experience with using silicone immersion objectives for regular pond life microscopy? I haven't seen this subject discussed much on here.

Silicone immersion objectives mostly seem to be used in research for imaging deep into living cells, as the refractive index of silicone oil (1.40) is very close to that of living cells (1.38).

However, it seems to me that silicone immersion objectives could also be useful for imaging live pond critters in water mounts? I guess most live pond critters also have a RI around 1.38, and the water has a RI of 1.33, so the silicone oil is much more similar to both of them than regular immersion oil (1.52). So I'm thinking it should behave a bit more similar to water immersion? Maybe silicone objectives would even work quite well when used with water as immersion medium?

Are there any other major drawbacks to silicone immersion objectives (except for their scarcity and price)? Is it really messy to clean up?

Hobbyst46
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Re: Silicone immersion objectives - any experience?

#2 Post by Hobbyst46 » Fri Jan 15, 2021 10:59 am

My occasional experience with low-density silicone oil (not for microscopy) is that it IS messy. On the other hand it is very stable, much more so than any natural oil. Will not deteriorate with time. And fairly expensive - since the smallest package is way larger to what we need for microscopy.
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Re: Silicone immersion objectives - any experience?

#3 Post by MicroBob » Fri Jan 15, 2021 11:20 am

Hi Vikor,
from the r.i. that sounds quite interesting for work on preparations with a thicker water layer. But silicone really is messy. It invisibly spreads over surrounding surfaces and interferes with wettability and adhesion. Car painters do hate it!
For workshop use thin silicone oil in spray cans is sold. But I stay away from it due to the dangers of contamination.
If one would absolutely want to use it one should define a process that limits contamination. E.g. use a dedicated microscope and work place for it.

Bob

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Re: Silicone immersion objectives - any experience?

#4 Post by viktor j nilsson » Fri Jan 15, 2021 11:31 am

Oh boy, that sounds even messier than I had expected. I definitely don't have room for another microscope and work place! It would need to go on the same microscope as my dry and oil objectives.

Could you explain a bit more how a drop of silicone oil on a slide would behave?

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Re: Silicone immersion objectives - any experience?

#5 Post by viktor j nilsson » Fri Jan 15, 2021 12:13 pm

Here's another thought. A 50/50 glycerol/water solution has a RI of 1.398 at 20°C. Maybe that could be a workable substitute for silicone oil? I think one of the reasons silicone is favored in research is that it's so stable, does not evaporate and that it's RI changes very little with temperature. But those things would less critical for me. Do you think glycerol/water would work well?

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Re: Silicone immersion objectives - any experience?

#6 Post by MicroBob » Fri Jan 15, 2021 12:41 pm

Oil can be removed fairly well by wiping with lighter fluid, so objective and slides are really clean. Silicone is quite inert and doesn't dissolve in usual cleaning fluids, so you push it from left to right and back to the left. A fine silicone layer or tiny drops of silicone oil repell water and other fluids (like car paint :? ).

Water-glycerin mix could work well as an immersion medium but the seal of the objective's front lens might not be tested for it. Depending on the price of the objective you could just try it. Water-glycerin mix shouldn't be very likely to cause damage. There are multi immersion objectives that can be used with water, oil or glycerin. Would that be an alternative?

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Re: Silicone immersion objectives - any experience?

#7 Post by viktor j nilsson » Fri Jan 15, 2021 1:28 pm

MicroBob wrote:
Fri Jan 15, 2021 12:41 pm
Oil can be removed fairly well by wiping with lighter fluid, so objective and slides are really clean. Silicone is quite inert and doesn't dissolve in usual cleaning fluids, so you push it from left to right and back to the left. A fine silicone layer or tiny drops of silicone oil repell water and other fluids (like car paint :? ).

Water-glycerin mix could work well as an immersion medium but the seal of the objective's front lens might not be tested for it. Depending on the price of the objective you could just try it. Water-glycerin mix shouldn't be very likely to cause damage. There are multi immersion objectives that can be used with water, oil or glycerin. Would that be an alternative?
Thanks! Silicone oil does sound awful in practice. One pdf from Nikon recommended petroleum benzine followed by pure ethanol to clean immersion oil, noting that the same could be used to clean silicone oil. It doesn't sound like it's quite that easy.

I've seen the Zeiss multi-immersion objectives, and those are likely great. But I'm looking at a specific objective, and I'm only really interested if the price is right. So the multi immersion objectives aren't really an option for me. I'll let you know how it goes.

I doubt that glycerin and water would be a problem for an objective that can keep silicone out, but of course your never know before you try.

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Re: Silicone immersion objectives - any experience?

#8 Post by Wes » Fri Jan 15, 2021 4:01 pm

viktor j nilsson wrote:
Fri Jan 15, 2021 12:13 pm
Here's another thought. A 50/50 glycerol/water solution has a RI of 1.398 at 20°C. Maybe that could be a workable substitute for silicone oil?
As far as refractive index goes it sounds fine but the mixture could differ in its dispersion properties relative to the oil.
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Re: Silicone immersion objectives - any experience?

#9 Post by apochronaut » Fri Jan 15, 2021 5:34 pm

I use a 63 x 1.0 glycerin immersion objective sometimes. It is a planachromat. I also have a 40X 1.0 oil planapo. When the 40X planapo is bumped to 60X with the correct 15X eyepieces, the image quality is superior to that of the 63X 1.0 glycerin. The glycerin objective does hold up to pushing with 15X eyepieces but always it's achromat heritage places limitations on it's usefulness.
Lately I have water immersion 1.10 objectives for the Nikon 200mm tube lens. Planachromats. I haven't fully evaluated them yet but I will try one with a glycerin/water mixture early next week. So too the 63X glycerin immersion.

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Re: Silicone immersion objectives - any experience?

#10 Post by viktor j nilsson » Fri Jan 15, 2021 7:42 pm

Wes wrote:
Fri Jan 15, 2021 4:01 pm
viktor j nilsson wrote:
Fri Jan 15, 2021 12:13 pm
Here's another thought. A 50/50 glycerol/water solution has a RI of 1.398 at 20°C. Maybe that could be a workable substitute for silicone oil?
As far as refractive index goes it sounds fine but the mixture could differ in its dispersion properties relative to the oil.
Been reading some about this, but haven't really found a definitive answer. What i do know is this:

Water and glycerol have very similar dispersion characteristics, with an Abbe number of 56.

According to the safety data sheet, Olympus silicone immersion oil is 100% Polydimethylsiloxane (PDMS).

I haven't found a definite source on the abbe number of PDMS, but according to some sources it's around 50. One source said 44, but I think that's incorrect.

If anyone knows where to find that information, let me know!

I also don't know how big of a difference 50 and 55 is. So... I'll just need to find out if I buy a silicone immersion objective...

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Re: Silicone immersion objectives - any experience?

#11 Post by viktor j nilsson » Sat Jan 16, 2021 3:31 pm

Well, I picked up the Olympus short-barrel 40x 1.00 Apochromat Silicone Immersion objective that I had been looking at. Got it for $125 and cheap shipping, which seemed like a reasonable gamble. I really like 40x immersion objectives, and the more I thought about it the more sense it made to use Water/Glycerine immersion for pond life. The short-barrel (non-plan) Apo40 0.85 is quite nice, so I don't think that the Apo40 1.00 will be too shabby either.

Really looking forward to seeing how it works with thick water mounts.

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Re: Silicone immersion objectives - any experience?

#12 Post by MicroBob » Sat Jan 16, 2021 8:16 pm

Hi Viktor,
please report how you get on with this very special objective. A 40:1 1.0 will be useful for many purposes. Do you know the free working distance to the cover slip?
Now it would be nice if you can find a 100:1 silicone immersion to accompany it! :lol:

Bob

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Re: Silicone immersion objectives - any experience?

#13 Post by viktor j nilsson » Sat Jan 16, 2021 8:32 pm

MicroBob wrote:
Sat Jan 16, 2021 8:16 pm
Hi Viktor,
please report how you get on with this very special objective. A 40:1 1.0 will be useful for many purposes. Do you know the free working distance to the cover slip?
Now it would be nice if you can find a 100:1 silicone immersion to accompany it! :lol:

Bob
I sure will report on it!

Haven't seen data for the SI Apo40 1.00 in any catalogues, but the Apo40 1.00 oil, which is identical except for the immersion medium, has a 0.19mm working distance. So it should be very close to that.

By the way, both Apo40 1.00 objectives also have an iris. So I think that it might produce some really nice high-NA darkfield images with my Heine.

Olympus did make a matching Non-fluorescent fluorite NA 1.30/100x Silicone oil immersion objective. If I find it, I would be tempted. The regular 100x 1.30 fluorite
oil is really good.

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Re: Silicone immersion objectives - any experience?

#14 Post by Scarodactyl » Sat Jan 16, 2021 10:49 pm

If nobody minds something of a diversion, I recently came across a weird silicone immersion objective:
https://www.ebay.com/itm/161285473379
Image
Feast your eyes on those specs! Those bizarre, baffling specs!

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Re: Silicone immersion objectives - any experience?

#15 Post by viktor j nilsson » Sat Jan 16, 2021 10:57 pm

:shock:

My eyes grew wider and wider the more i looked at that picture. I don't understand how that would even be possible. Do you know anything about it? Can't find any information online.

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Re: Silicone immersion objectives - any experience?

#16 Post by hans » Sat Jan 16, 2021 11:14 pm


Hobbyst46
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Re: Silicone immersion objectives - any experience?

#17 Post by Hobbyst46 » Sat Jan 16, 2021 11:27 pm

Very interesting. Indeed SIL (solid imm.) yields super resolution; there is a lens that is made of high refractive material. Now must read the article to fully understand. Thanks !
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Re: Silicone immersion objectives - any experience?

#18 Post by viktor j nilsson » Sat Jan 16, 2021 11:36 pm

That makes a lot more sense now, thanks.

By the way, circling back to silicone (and water/glycerol) immersion, I thought it would be nice to put this figure here as it illustrates very well why I became interested in trying silicone immersion objectives with pond life:

Image

I think it applies to live cell imaging (through cell tissues with RI=1.38), but the overall picture should be similar when focusing through a water mount (RI=1.33). In fact, the oil objective would be even more at a disadvantage in water. But the water immersion objective would fare a bit better.

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Re: Silicone immersion objectives - any experience?

#19 Post by MichaelG. » Sun Jan 17, 2021 12:20 am

This is way out of my league, Viktor ... but may be of interest:
https://www.olympus-lifescience.com/en/ ... l-imaging/

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Re: Silicone immersion objectives - any experience?

#20 Post by viktor j nilsson » Sun Jan 17, 2021 7:39 am

Thanks MichaelG. Yes, silicone immersion is clearly very useful when trying to reconstruct what's going on deep inside cells and seems to be increasingly used in super resolution fluorescence microscopy. I have some plans to build a DIY structured illumination super resolution microscope at some point, the non-fluorescing silicone objectives could turn out to be useful for that. Would be kind of cool to use a late-70's objective for that!

I have been a little curious about the history of the Olympus silicone objectives. They are listed in a price list from 1978:
http://www.alanwood.net/downloads/olymp ... scopes.pdf

The Olympus silicone objectives seem relatively common on the used market. But I don't think I've ever seen any Nikon silicone immersion objectives from the same 1970-1980's era (short barrel S series, long barrel CF series), which has puzzled me a bit.

But then I found this 1976 Olympus Corp. patent:
https://patents.google.com/patent/US4108794A

In it, Olympus simply patents the use of pure silicone oil as an immersion medium. So this was clearly a new thing then. The patent does not discuss the RI of silicone oil as an advantage that lets you see deeper into cells. Rather, it highlights the fact that silicone oil is non-fluorescent, does not dry out and that it has a stable RI that is "relatively close to that of cargille oil", which is portrayed as having the ideal RI. (The low RI of distilled water is described as a limitation).

Interestingly, the patent also describes how regular oil immersion objectives can be tweaked for silicone immersion by moving the second lens group slightly.

From this, it seems like Olympus was trying to create a market for silicone immersion objectives at around this time (1976-1980), and were probably quite alone in doing so. But that the driver behind it was mainly that there didn't really exist other good, non-fluorescing immersion oils at the time. When non-fluorescens oils arrived a couple of years later, I guess that the demand for silicone immersion objectives went down. Until modern live-cell imaging techniques increased the demand for objectives that could see deeper into living tissue.

At least that's my current (limited) understanding of the historical context!

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Re: Silicone immersion objectives - any experience?

#21 Post by MichaelG. » Sun Jan 17, 2021 9:12 am

.
Thanks for sharing your thinking [and also the patent link], Viktor
... please keep us informed of your progress!

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Re: Silicone immersion objectives - any experience?

#22 Post by Hobbyst46 » Sun Jan 17, 2021 9:49 am

Thanks Michael and Viktor,
Incidentally, I am sure I have once seen an old 100X objective - almost certainly Olympus - that bore the mark "SI". In retrospect, might have been short for Silicone Immersion ?
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Re: Silicone immersion objectives - any experience?

#23 Post by viktor j nilsson » Sun Jan 17, 2021 12:48 pm

Hobbyst46 wrote:
Sun Jan 17, 2021 9:49 am
Thanks Michael and Viktor,
Incidentally, I am sure I have once seen an old 100X objective - almost certainly Olympus - that bore the mark "SI". In retrospect, might have been short for Silicone Immersion ?
Yup, those are the short barrel silicone oil immersion objectives.

The price list from 1978 has a FL100 1.30 SI objective listed, but I haven't been able to find any images of that one. The only 100x ones I've found have been labelled FL100 1.25F SI. They seem to have been rather common. Maybe Olympus realized that 1.30 was a but optimistic and lowered the NA when it went into production? Or maybe the 1.30 was a later updated version.

Anyway, I just bought one of the FL100 1.25 SI objectives for $44, so I'll be able to report on that one in due time.

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Re: Silicone immersion objectives - any experience?

#24 Post by hans » Sun Jan 17, 2021 7:01 pm

viktor j nilsson wrote:
Sat Jan 16, 2021 11:36 pm
By the way, circling back to silicone (and water/glycerol) immersion, I thought it would be nice to put this figure here as it illustrates very well why I became interested in trying silicone immersion objectives with pond life:
Have you come across any diagrams like this with more explanation, like: Are the values measured, simulated, or calculated from some approximation? Does the behavior vs. depth depend a lot on the exact design of the objective or is it similar across typical designs?

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Re: Silicone immersion objectives - any experience?

#25 Post by viktor j nilsson » Sun Jan 17, 2021 10:38 pm

hans wrote:
Sun Jan 17, 2021 7:01 pm
viktor j nilsson wrote:
Sat Jan 16, 2021 11:36 pm
By the way, circling back to silicone (and water/glycerol) immersion, I thought it would be nice to put this figure here as it illustrates very well why I became interested in trying silicone immersion objectives with pond life:
Have you come across any diagrams like this with more explanation, like: Are the values measured, simulated, or calculated from some approximation? Does the behavior vs. depth depend a lot on the exact design of the objective or is it similar across typical designs?
I don't know how that figure was produced, unfortunately. It comes from olympus, but they haven't provided any real detail anywhere that I can find. It does look kind of stylized.

But from some examples I've found, the differences between oil and water/silicone seem pretty striking when used for live cell imaging.

The Scaleview-2 example on the following page shows slices taken at different depth, showing striking differences in brightness:
https://www.olympus-lifescience.com/en/ ... l-imaging/

And the following figure nicely show differences in the ability to resolve structures at different depths between oil and water immersion, but sadly doesn't show silicone oil:
https://www.researchgate.net/figure/Com ... _324903972

I would think that these results are fairly general, and not limited to a specific design. The deterioration with depth with oil is a very simple effect of spherical aberration arising from the RI mismatch. I think that the optics involved in that must have been well understood by the 1960s. So switching from an objective optimized for oil, with an RI of 1.52, to an objective optimized for silicone oil, with an RI 1.40, will likely have a much bigger effect (when looking at things with a RI of 1.38) regardless of the exact design of the objective.

While modern objectives have incorporated some very fancy things, like motor-controlled correction collars that can compensate for small differences in RI due to temperature and evaporation, I believe that those improvements are going to be relatively small compared to the overall effect of more closely matching the RI of the immersion medium and the sample.

But whether 'my' 1970s silicone objectives turn out to be useful for taking pretty pictures of protists in wet mounts remain to be seen!

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Re: Silicone immersion objectives - any experience?

#26 Post by viktor j nilsson » Wed Jan 20, 2021 7:03 am

Just adding some additional information for completeness.

Olympus did make at least some silicone immersion objectives during the BH-2 era. According to Carl Hunsingers's documents, there was 100x 1.20 SI achromat, a UV fluorite 40x 1.30 SI phase contrast objective, and a UV Fluorite 100x 1.30 SI phase contrast objective.

They look like this:
L2.jpeg
L2.jpeg (64.81 KiB) Viewed 1787 times
L1.jpg
L1.jpg (107.1 KiB) Viewed 1787 times
I find it a little weird that they were phase contrast. I did find at least one paper from the 1990s that used the UVFL40xPL Sil for fluorescence microscopy. I guess phase contrast helped with viewing cells before switching on the epi fluorescence unit, and that the phase rings weren't seen as being too detrimental for image quality when capturing the fluorescence emission.

http://e-micro.jp/actually has both of them for sale (images above are from their listing). I would think that they were very rare. They are not documented in the mainstream catalogues, but where likely only advertised in more specialized literature.

None of the Nikon experts have heard about any Nikon silicone immersion objectives from the finite era. I think it's safe to say that Nikon did not make any until very recently.

Leica and Zeiss were likely quite satisfied with their multi-immersion objectives, and still are.
Last edited by viktor j nilsson on Wed Jan 20, 2021 7:08 am, edited 2 times in total.

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Re: Silicone immersion objectives - any experience?

#27 Post by viktor j nilsson » Wed Jan 20, 2021 7:07 am

s-l400.jpg
s-l400.jpg (17.14 KiB) Viewed 1787 times
i-img1200x900-15996585548ceo5v3654610.jpg
i-img1200x900-15996585548ceo5v3654610.jpg (42.2 KiB) Viewed 1787 times
And here are the two short-barrel (BH era) silicone objectives that are currently on their (long) way to me.

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Re: Silicone immersion objectives - any experience?

#28 Post by Hobbyst46 » Wed Jan 20, 2021 9:15 am

A 100X1.25 SI objective like the one shown in the bottom photo must be in one of my drawers. If I can find it, attach to the DIN micoscope via a spacer ring to create some parfocality, and get some silicon oil, then it will be interesting to test.

Viktor - which specimen do you plan to image with the silicon objective ? hopefully not only live cells ? and not just a fluorescent material ? because neither of these are available to me.
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Re: Silicone immersion objectives - any experience?

#29 Post by viktor j nilsson » Wed Jan 20, 2021 9:40 am

Hobbyst46 wrote:
Wed Jan 20, 2021 9:15 am
A 100X1.25 SI objective like the one shown in the bottom photo must be in one of my drawers. If I can find it, attach to the DIN micoscope via a spacer ring to create some parfocality, and get some silicon oil, then it will be interesting to test.

Viktor - which specimen do you plan to image with the silicon objective ? hopefully not only live cells ? and not just a fluorescent material ? because neither of these are available to me.
Would love to hear how that works for you! I think the tube length error will be problematic at that high NA. If you are able to focus without a spacer, it would be good to do that at least for testing purposes.

My plan is to use these objectives for viewing and photographing live protists (with DIC) in relatively deep water mounts. My thinking is that, since silicone has a refractive index much closer to water (1.40 vs 1.33) than oil (1.52), they will allow me to photograph deeper into my water mounts (and inside protists) while maintaining decent resolution and minimizing spherical aberration than I could do with oil immersion objectives. By varying the water:glycerine concentration, I might even be able to compensate for the refractive mismatch to some extent (similar to using #1 cover glass with oil immersion). I'm more into temporary water mounts than permanent mounts, so I am rarely able to take full advantage of oil immersion.

So, basically I plan to use them as I would use water immersion objectives. But Olympus didn't make any short-barrel water immersion objectives.

At this point, I've gathered quite an extensive collection of high-end short-barrel objectives for my Vanox AH. I recently took delivery of a 20x planapo, which turned out to give considerably better DIC effect with my 40x DIC prism than my long-barrel Nikon 20x objectives. That sort of sealed the deal for me, as I have previously been on the fence of whether I should stick to the original short-barrel Olympus objectives, or use my Nikon objectives on the Vanox. I think that I am now committed to the old-but-good short-barrel Olympus objectives. And since the silicone immersion objectives are from the same era, it made sense to add them to the mix and see what they can do.
Last edited by viktor j nilsson on Wed Jan 20, 2021 11:03 am, edited 2 times in total.

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Re: Silicone immersion objectives - any experience?

#30 Post by viktor j nilsson » Wed Jan 20, 2021 9:43 am

Hobbyst46 wrote:
Wed Jan 20, 2021 9:15 am
A 100X1.25 SI objective like the one shown in the bottom photo must be in one of my drawers. If I can find it, attach to the DIN micoscope via a spacer ring to create some parfocality, and get some silicon oil, then it will be interesting to test.
If you don't want to mess with silicone oil, I really think that a 50:50 water:glycerine mixture will work really well, and be a lot easier to clean. I doubt that it will damage the objective in any way (unless the seal is already damaged).

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