I'm beginning to think oil-immersion isn't worth it...

Beatsy · Post by **Beatsy** » Thu Jul 20, 2017 2:03 am

...because water immersion is plenty good enough in the overwhelming majority of cases.

I've recently been stacking diatoms using a 40/0.9 multi-immersion objective on the "water" setting in conjunction with a dry N.A. 1.4 condenser (effectively N.A. 1.0). I was about to switch to a 63/1.4 oil immersion objective and oil the condenser for better resolution, but a quick calculation pretty much demonstrated it wouldn't be worth it. Very roughly, the WI objective resolves to 300nm while the oiled objective and condenser would only improve that to 220nm - with a massive amount of extra faffing and cleaning added to the process.

The lazy slug in me is much preferring the water option

Ichthyophthirius · Post by **Ichthyophthirius** » Thu Jul 20, 2017 3:59 am

Hi,

If you're in the enviable position to have a high NA water immersion objective

It's a good idea, though, to do water immersion of the condenser front lens as well. It really brightens things up.

Regards, Ichty

Beatsy · Post by **Beatsy** » Thu Jul 20, 2017 4:08 am

Ichthyophthirius wrote:It's a good idea, though, to do water immersion of the condenser front lens as well. It really brightens things up.

I hadn't thought of that - extra light is often welcome. Thanks Ichty.

zzffnn · Post by **zzffnn** » Thu Jul 20, 2017 6:58 am

Beatsy,

Did your 220nm vs 300nm resolution difference came from comparing 63/1.4 oil and 40/0.9 water? Did your calculation include degradation by SA?

Please note some condensers are not sealed against water. I have been water-immersing my LOMO Abbé 1.25 condenser for a year and water leaked beneath its top lens. It was easy to clean up and repair with glass cement though, since it is a simple 2-lens modular design. And it costs less than $30 to replace.

An 1.4 NA condenser may not be so easy to repair? I certainly won't immerse my Leitz Heine with water. I won't use glycerine either, as it gets mold easily (I wipe it off every time, but still).

You would guess since LOMO, Zeiss and Leitz all made water immersion objectives, their condensers should be water proof. But apparently my LOMO Abbé is not.

Ichthyophthirius · Post by **Ichthyophthirius** » Thu Jul 20, 2017 7:35 am

zzffnn wrote:Did your 220nm vs 300nm resolution difference came from comparing 63/1.4 oil and 40/0.9 water? Did your calculation include degradation by SA?

Hi,

The Zeiss 40/0.9 multiimmersion objective has a correction ring all the way from water to oil so there is scope to reduce SA this way.

Maybe the issue was with the Lomo condenser rather than the water immersion? It seems unlikely that a condenser was built with water-soluble lens cement. If the water was ultrapure, it should just evaporate without leaving any residue behind.

Regards, Ichty

Beatsy · Post by **Beatsy** » Thu Jul 20, 2017 7:39 am

zzffnn wrote:Did your 220nm vs 300nm resolution difference came from comparing 63/1.4 oil and 40/0.9 water? Did your calculation include degradation by SA?

Yes but no allowance for SA (don't know how to calculate for that anyway). It was just a rough approximation based on wavelength/(2NA).

Please note some condensers are not sealed against water.

My Zeiss condenser has a separate screw on top lens for NA 1.4. Designed for immersion and no liquid touches the condenser itself - only this top lens.

Just to clarify what I meant by "not worth it". Sure, there will be times when a water immersion objective can't quite resolve the dots in a diatom (say) and the striae just appear grainy or "moniliform" (lovely word). Under those circumstances, using oil and a higher NA objective may separate the dots. But for most diatoms, just detecting dots is sufficient; there's no need to fully resolve them (they'll still only be dots, albeit better separated ones). I'm generally content with such a compromise if it avoids onerous cleaning chores. Like lightning, I'll always take the path of least resistance

Choronzon · Post by **Choronzon** » Thu Jul 20, 2017 11:30 am

Well, several things about oil immersion. I does give the best resolution, but also causes the best specimen disruption on everything except fixed slides. With the multi immersion lenses, I've found that refractive index matching is where they excell. My issue with oil immersion stems from cover-slipped slides from my "tank of death", which is my cryptobiotic fish tank that is going on its 4th year of desiccation / rehydration. To see good detail in the rehydrated organisms like rotifers, I need about 40 to 50x magnification. Because of the floating temporary mounts of the upright dic microscope configuration, oil immersion displaces the coverslip in a disastrous way in vivo.
The 50x Leitz PL Fluotar *1.0 that I recently found, solves that issue, as well as any spherical aberration from refractive index mismatch. So for live imaging of organisms, for me, water immersion is the only way to go.

Cactusdave · Post by **Cactusdave** » Thu Jul 20, 2017 2:27 pm

There's no doubt that multi immersion objectives are very nice, but they are eye wateringly expensive and rare compared to conventional oil immersion objectives. Even a good delamination-free Zeiss X63 planapo, 1.4 oil, to me pretty much the gold standard high resolution, high magnification 160mm objective, will only cost you a fraction of the secondhand price of any of the Zeiss 160mm multi immersion objectives, when they ever appear.

Choronzon · Post by **Choronzon** » Thu Jul 20, 2017 8:26 pm

One needs to be astute on eBay. And let's face facts, this still is the market. I recently bought a Zeiss 16X multi immersion lens on eBay for $200. If that's too much for your hobby there is no hope. The risk is delamination with Zeiss objectives, but they're worth the hunt.

zzffnn · Post by **zzffnn** » Fri Jul 21, 2017 6:41 am

Choronzon wrote:One needs to be astute on eBay. And let's face facts, this still is the market. I recently bought a Zeiss 16X multi immersion lens on eBay for $200. If that's too much for your hobby there is no hope. The risk is delamination with Zeiss objectives, but they're worth the hunt.

That is a great deal! Normally that kind of multi-immersion objectives go north of $400, mostly over $600-800.

Any secrets for other eBayers? Uncommon search terms, misspell search, use "others also viewed", or something else? Thank you for sharing.

GaryB · Post by **GaryB** » Fri Jul 21, 2017 4:56 pm

I'm in the same boat, oil is just too messy and impractical for me! I've been thinking of water immersion but wondered what people normally do about contamination with, say, salt water specimens under a cover slip and non saltwater immersion. If the edge of the slip is reached (as I frequently do) the two waters will inevitably cross contaminate. Is this a problem and does it mess up the view? I'm looking to pull the trigger on a WI objective but am unsure as I have no experience with them.

One worry is excess water where everything starts to wobble with the slightest movement.

Ichthyophthirius · Post by **Ichthyophthirius** » Fri Jul 21, 2017 5:31 pm

Hi Gary,

There are several solutions. Sample preparation is the key.

Normally, you coverslip your sample and then remove all excess water with a paper tissue. The remaining water layer is so thin that the coverslip won't move any more!

You can further improve the mount by using larger cover glasses (e.g. 22 x 40 mm).

If you can't remove all excess water (because the cover glass crushes the sample), leave more water but seal the edges off with nail varnish. Even with immersion oil, the cover glass won't move any more.

If very thick mounts are necessary, you can support the cover glass with other cover glasses to make a chamber: http://www.bio.brandeis.edu/marderlab/c ... rslips.jpg

The surface tension of both water and immersion oil is high enough so it stays on top of the cover glass and won't mix with teh sample if you do the preparations this way.

Another solution is the use of low-viscosity immersion oil to reduce the drag on the cover glass.

Regards, Ichty

zzffnn · Post by **zzffnn** » Fri Jul 21, 2017 5:43 pm

GaryB,

I would suggest that you avoid water immersion objectives with NA of less than 0.9, if you use cover slips. Those low NA water objectives are mostly used for no cover dipping. For example, LOMO water 40x NA 0.75 has such a huge working distance, that immersion water (needed to bridge gap) may flood your covered sample.

I also use as minimal immersion water as possible and use a super fine tip pipette to deliver just enough immersion water.

Towards the very edge of your covered sample, flooding and cross contamination is hard to avoid, unless you seal your cover slip with immersion oil or nail varnish. I don't like to seal my cover slips, so I only use a high dry (40x NA 0.95) objective at edges and I always image with very thin water samples.

Ichthyophthirius · Post by **Ichthyophthirius** » Fri Jul 21, 2017 5:45 pm

zzffnn wrote:Normally that kind of multi-immersion objectives go north of $400

Hi,

Don't worry; the WD for the 16x is just 0.15 mm (a bit longer with water, I suppose). When would you ever use it? At such a working distance, you have to prepare a very thin mount anyway and if you do that, you can just use a dry objective or an oil immersion. These objectives were intended for fluorescence and not so much for pond objects.

Compared for example with the Nikon CF Fluor 10/0.5, which has a working distance of 0.88 mm!

Regards, Ichty

zzffnn · Post by **zzffnn** » Fri Jul 21, 2017 6:00 pm

Ichty,

I was just commenting on multi immersion objectives in general, not specifically for the 16x objective. Yes, most people may not use low power oil immersion that much. I do use a 11x 0.25 and a 30x 0.65 oil objective for scanning, when I lose my live protists at 60- 90x oil (no need to clean up immersion oil that way). High NA provided by immersion, as you pointed out, is another application.

Dipping into deep pond sample to image bottom ameobas or diatoms (or any bottom protists that do not swim up and down), is a yet another application for low NA water immersion.

I would guess that at immersion NA of 0.4 or less, one may use either water or oil interchangeably for the same objective, assuming such objective is sealed against water. Not sure if one can push that immersion interchangeability up to NA 0.65 though (would be nice to know).