"Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by Pau »

You can try oil immersion between the slide and the cover if your test slide can resist the oil and cleaning process.

Anyway the visible difference will not be high if any, specially with the 1.25 achro
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viktor j nilsson
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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by viktor j nilsson »

At least for me, it would really help to see a wider FOV, I'm not used to seeing only a 400% crop. I've also been quite unimpressed with 100x 1.25 achromats in the past (like my Nikon E plan) so I'm not overly surprised to see CA in the centre, but it's just not clear to me exactly how pronounced it really is.

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Re: Disastrous 100x oil immersion objective comparison

Post by Duke »

Pau wrote:
Sat Sep 12, 2020 9:07 am

Hi Duke, this has been discussed in a now 6 years old thread
viewtopic.php?p=139486#p139486

In fact there are objectives marked "no cover glass" or "../0" not only dry but also (rare) oil immersion ones meant for smears
There's a multiple studies of this phenomenon in scientific literature, both theoretical and practical, which showed the fact that coverglass thickness won't matter on PSF of oil immersion objectives, as long as all conditions are optimal (oil immersion optical properties are highly influenced by environment, such as temperature and atmospheric pressure).
Such markings are necessary, but for a different reason. Mainly, because the working distance of the objectives would not be right, for the different slip thickness. Immersion objectives marked ../0 wouldn't even allow use of the cover glass, because their working distance is on the magnitude of 0.20mm, therefore it tells using it with cover glass it will only result in damaging front element.
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Ichthyophthirius
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Re: Disastrous 100x oil immersion objective comparison

Post by Ichthyophthirius »

Duke wrote:
Sun Sep 13, 2020 1:50 am
There's a multiple studies of this phenomenon in scientific literature, both theoretical and practical, which showed the fact that coverglass thickness won't matter on PSF of oil immersion objectives, as long as all conditions are optimal (oil immersion optical properties are highly influenced by environment, such as temperature and atmospheric pressure).
Hi,

I disagree with that part. It's important to note that there is an NA mismatch between coverglass (1.522) and oil (1.518). Therefore, the PSF will be affected by cover glass thickness at high NA.

At low NA (1.25) it doesn't matter if a cover glass is used.

At high NA (e.g. 1.4), because of the NA mismatch, the PSF is affected by cover glass thickness.

Very high NA objectives (e.g. Leica 63x/1.47) even have correction collars for cover glass thickness (at a given temperature). As you mentioned, the temperature (23 vs. 37C) has a big influence on the NA mismatch at very high NA.

I think this is the paper that showed the influence of the cover glass thickness on the PSF. Unfortunately I don't have access any more (it used to be on the authors university server but is not available any more): https://www.osapublishing.org/josaa/abs ... aa-9-1-154 Sarah Frisken Gibson and Frederick Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 9, 154-166 (1992)

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Re: Disastrous 100x oil immersion objective comparison

Post by Duke »

Ichthyophthirius wrote:
Sun Sep 13, 2020 3:49 am
I think this is the paper that showed the influence of the cover glass thickness on the PSF. Unfortunately I don't have access any more (it used to be on the authors university server but is not available any more): https://www.osapublishing.org/josaa/abs ... aa-9-1-154 Sarah Frisken Gibson and Frederick Lanni, "Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy," J. Opt. Soc. Am. A 9, 154-166 (1992)
From this paper p. 157
These sources include the use of a coverslip with a thickness or refractive index differing from that of the design system, the use of a nondesign immersion oil, the defocus of the object such that its Gaussian image point is shifted from the detector plane, the location of the detector at a nondesign detector plane, and the immersion of the point source into a medium whose refractive index is significantly different from that of the immersion oil. According to this model, the aberration is independent of the order of the layers separating the object plane from the front element of the objective lens. Consequently, if the refractive index of the specimen is equal to that of the immersion oil, the contribution to the OPD, which depends on the depth of the point source into the specimen layer, is 0.
p. 162
Figure 9 illustrates the dependence of the PSF on the thickness of the coverslip when other parameters meet the microscope design conditions. It can be seen in this figure that, for an oil-immersion objective lens, the OSM PSF is relatively independent of coverslip thickness. By using Eq. (5) it can be shown that a change in thickness of the coverslip of ±30 m from the design thickness results in less than 1/4 wavelength of spherical aberration added to the image. Figure 10 shows cross sections of measured PSF's of the OSM microscope with microspheres embedded in optical cement immediately below a 0.170-mm-thick coverslip for immersion oils with refractive indices varying from 1.500 to 1.530 about the design refractive index, 1.515. There is little effect on the OSM PSF for a difference in the refractive index from that of the design immersion oil of less than 0.005.
So, when using a oil-immersion without cover glass directly on the thin sample : 1. there's no optical path difference (OPD ~ 0). 2. Using the formulas aberration results in less than 1/4 λ.
In such case objective is actually much more close to ideal designed scenario, than in most of the cases of it's typical use, when object is mounted to medium with refractive index much different from designed 1.520.
“Thoroughly conscious ignorance is the prelude to every real advance in science.” - JCM

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Re: Disastrous 100x oil immersion objective comparison

Post by Macro_Cosmos »

Duke wrote:
Sun Sep 13, 2020 11:45 pm

From this paper p. 157

So, when using a oil-immersion without cover glass directly on the thin sample : 1. there's no optical path difference (OPD ~ 0). 2. Using the formulas aberration results in less than 1/4 λ.
In such case objective is actually much more close to ideal designed scenario, than in most of the cases of it's typical use, when object is mounted to medium with refractive index much different from designed 1.520.
Interesting, I'd love to have a look at the paper.
Your location is interesting too... :mrgreen:

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Re: Disastrous 100x oil immersion objective comparison

Post by Duke »

Macro_Cosmos wrote:
Mon Sep 14, 2020 3:24 am
Interesting, I'd love to have a look at the paper.
Your location is interesting too... :mrgreen:
You see, as I would like to share it, but, according to publishing legal statements, it'd be a copyright infringement, so site as well as me might get in trouble. I suspended my PhD in chemistry, but still have access to papers.
However, and regarding the location, you should know, that soviet upbringing had axioms burned deep in our minds, such as "public availability of the knowledge and education", that many of us will never give up. Some of such people, overwhelmed with frustration of "unfair capitalistic world", openly crusade against the system, in such act, sharing public access to all scientific papers thru their site (yes, it's up for many years, and is still available for everyone, free to use).
I, personally, do not endorse "piracy" and "hacking", but, I do understand their motivation.
“Thoroughly conscious ignorance is the prelude to every real advance in science.” - JCM

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by adamski »

Hi,

I did my own comparison, same setup but different objectives 400% centre crop.

From left to right:
UplanFL 100x 1.30 PH3 UIS, UplanApo 100x 1.30 Iris UIS, UplanSapo 1.40 UIS2
100x test.jpg
I have not done any adjustments, straight from the camera. UplanSapo destroys both as far as contrast and clarity, especially when you look through oculars. That's why they cost the money :)

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by Macro_Cosmos »

adamski wrote:
Tue Sep 15, 2020 9:41 am
I have not done any adjustments, straight from the camera. UplanSapo destroys both as far as contrast and clarity, especially when you look through oculars. That's why they cost the money :)
Good to know! Yeah, the 100x UplanSApo just obliterates the competition. I'd say it's consistent with my results in this case.

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by pbraub »

I share your impression that there is not much difference between the semi-apos and apos of olympus (especially the newer lines - (U)PLanFl N and UPlanSApo. The jump from achromats to flourites is obvious however (i use fl and apos on my diagnostic scope and would never go back).

Some while ago i had the chance to test out the new olympus apo line (UPlanXApo). It completely blew my mind. With some pixel-peeping you can still see some lateral chromatic abberation on high contrast objects (phagocytosed dust particles in macrophages in my case) when zooming. With the XApo there was NONE - and a crystal clear overly sharp impression during observation. Do I need this, probably not -- wanting however is another topic :D

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by dolmadis »

pbraub wrote:
Wed Sep 16, 2020 8:02 am
I share your impression that there is not much difference between the semi-apos and apos of olympus (especially the newer lines - (U)PLanFl N and UPlanSApo. The jump from achromats to flourites is obvious however (i use fl and apos on my diagnostic scope and would never go back).

Some while ago i had the chance to test out the new olympus apo line (UPlanXApo). It completely blew my mind. With some pixel-peeping you can still see some lateral chromatic abberation on high contrast objects (phagocytosed dust particles in macrophages in my case) when zooming. With the XApo there was NONE - and a crystal clear overly sharp impression during observation. Do I need this, probably not -- wanting however is another topic :D

What are are the semi apo olympus microscope objectives please?

Thanks

John

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by Pau »

Semi-apo refers to fluorites
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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by Macro_Cosmos »

pbraub wrote:
Wed Sep 16, 2020 8:02 am
I share your impression that there is not much difference between the semi-apos and apos of olympus (especially the newer lines - (U)PLanFl N and UPlanSApo. The jump from achromats to flourites is obvious however (i use fl and apos on my diagnostic scope and would never go back).

Some while ago i had the chance to test out the new olympus apo line (UPlanXApo). It completely blew my mind. With some pixel-peeping you can still see some lateral chromatic abberation on high contrast objects (phagocytosed dust particles in macrophages in my case) when zooming. With the XApo there was NONE - and a crystal clear overly sharp impression during observation. Do I need this, probably not -- wanting however is another topic :D
XApos! I'd love to try out a set, it's said they are corrected in 4 wavelengths?
I'd also love to try out silicone oil immersion objectives, they are however expensive and the immersion oil is very expensive too, I was quoted $200 a bottle.

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by Ichthyophthirius »

Macro_Cosmos wrote:
Thu Sep 17, 2020 12:55 am
I'd also love to try out silicone oil immersion objectives, they are however expensive and the immersion oil is very expensive too, I was quoted $200 a bottle.
You could try matching the RI by using a glycerol solution (51 % at 20C) although dispersion will probably be different between glycerol and silicone immersion oil http://edge.rit.edu/edge/P13051/public/ ... 0water.pdf

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Re: "Disastrous" (completely unexpected!!) 100x oil immersion objective comparison

Post by viktor j nilsson »

I know that Håkan Kvarnström, hkv, has laid his hands on some UPlanXApo's and posted some images on his instagram (@micromundusphotography). Not that his photos were lacking before, but the images from the XApos look outstanding.

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