Glycerol Immersion Objectives

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LouiseScot
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Glycerol Immersion Objectives

#1 Post by LouiseScot » Tue Apr 06, 2021 7:43 pm

Hi all

I understand glycerol immersion objectives are intended to work with relatively low refraction media e.g. glycerol/water Anyone had any experience with glycerol immersion? I'm thinking glycerin objectives could still work ok with other media of similar RI?

Thanks for any wisdom :)

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

apochronaut
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Re: Glycerol Immersion Objectives

#2 Post by apochronaut » Tue Apr 06, 2021 8:04 pm

I sometimes use a 63X 1.0 N.A. glycerin immersion planachro. It is very good but since I don't also have the compatible 100X 1.25 glycerin immersion planachro, it is a bit cumbersome. There are very few objectives that can be used with multiple immersion media, without having adjustment collars. This one needs glycerin, water isn't good enough, nor is oil.
Since I also have a 40X 1.0 N.A. planapo oil immersion objective, I find myself using it with 15X wide field eyepieces , which provides me with a better image at the equivalent of a 60X and has the added bonus of working nicely with any of the oil immersion 100X options.....particularly for DF.

LouiseScot
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Re: Glycerol Immersion Objectives

#3 Post by LouiseScot » Tue Apr 06, 2021 8:31 pm

apochronaut wrote:
Tue Apr 06, 2021 8:04 pm
I sometimes use a 63X 1.0 N.A. glycerin immersion planachro. It is very good but since I don't also have the compatible 100X 1.25 glycerin immersion planachro, it is a bit cumbersome. There are very few objectives that can be used with multiple immersion media, without having adjustment collars. This one needs glycerin, water isn't good enough, nor is oil.
Since I also have a 40X 1.0 N.A. planapo oil immersion objective, I find myself using it with 15X wide field eyepieces , which provides me with a better image at the equivalent of a 60X and has the added bonus of working nicely with any of the oil immersion 100X options.....particularly for DF.
Ok, thanks Apo. I suppose potential spherical aberration is the enemy here, and is probably why glycerin immersion objectives are relatively cheap...
If I bought one it would just substitute for my 100x oil immersion which I don't use very often anyway. The one I'm looking at is specified for uv, is 100x/1.30 and has an aperture collar: 0.8-1.30. though I couldn't properly use it at 1.30 anyway. 0.9-1.2 would be fine by me! I've obviously never used glycerin immersion before so not sure of the limitations. Ordinary cover slip glass might be a problem? Also, may not be that easy to find mounting media with a compatible RI?

Cheers

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

apochronaut
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Re: Glycerol Immersion Objectives

#4 Post by apochronaut » Wed Apr 07, 2021 12:52 am

Quartz cover slips might be of some advantage but the conventional suppliers want a pretty penny for them. I just happen to have some tweaked water immersion objectives coming from China soon and they are sending some quartz covers and slides at the same time. I am not sure about the price but it will likely be about 1/10 what the microscope supply companies want for them.

So, I will have some quartz glass available soon.

LouiseScot
Posts: 488
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Re: Glycerol Immersion Objectives

#5 Post by LouiseScot » Wed Apr 07, 2021 1:13 am

apochronaut wrote:
Wed Apr 07, 2021 12:52 am
Quartz cover slips might be of some advantage but the conventional suppliers want a pretty penny for them. I just happen to have some tweaked water immersion objectives coming from China soon and they are sending some quartz covers and slides at the same time. I am not sure about the price but it will likely be about 1/10 what the microscope supply companies want for them.

So, I will have some quartz glass available soon.
Hi again!

Nice to know though the price (each!) seems to be crazy... I've not yet managed to find out much about glycerine immersion - I suppose it's a bit niche. I can't find out much about the particular objective either. It's not looking such a good idea now unless I could get away with standard glass and a reduced aperture.

Cheers

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: Glycerol Immersion Objectives

#6 Post by Hobbyst46 » Thu Apr 08, 2021 8:39 am

Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

LouiseScot
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Re: Glycerol Immersion Objectives

#7 Post by LouiseScot » Thu Apr 08, 2021 9:45 am

Hobbyst46 wrote:
Thu Apr 08, 2021 8:39 am
Louise
This might be relevant
https://www.microbehunter.com/microscop ... ne#p101304
Hiya - thanks though the 100x/1.30 (0.8-1.30) objective I mentioned (a Nikon UV-F100) is specified for glycerine immersion. It is, however, really an epifluorescence / UV objective. Maybe at visible wavelengths it will be more forgiving? I hesitated, but in the end I bought it anyway! :) . I may have problems with it because of the need for low refractive index glass and media. Still, it will give me something else to play with and compare :D

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

LouiseScot
Posts: 488
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Re: Glycerol Immersion Objectives

#8 Post by LouiseScot » Thu Apr 08, 2021 10:58 am

I found this paper comparing glycerine and water but done theoretically and using an oil immersion lens NA = 1.30 as a theoretical reference, and for confocal fluorescence microscopy of thick specimens.

https://onlinelibrary.wiley.com/doi/pdf ... 0.CO%3B2-7

It's interesting, but a more practical investigation would be better! I'll keep looking!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

apochronaut
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Re: Glycerol Immersion Objectives

#9 Post by apochronaut » Thu Apr 08, 2021 12:37 pm

As with so many things , there is theory and then there is empirici.
There does seem to be a bit of a phobia that plagues newly hatched microscopists when it comes to oil immersion. It is a little difficult to master but immersion is immersion and outside of an extra minute or two to clean up a bit of oil, the intricacies are pretty similar between the various immersion media but the results different. Oil has the advantage though, of being much more common, so the glass being utilized on a broad scale for preparation is more homogeneous with oil, more widely available and cheaper.
Between the categories of objective colour correction , stepping around the immersion debate, the results speak for themselves. If one is content to gaze at pretty colours and shapes, then working at lower N.A. for convenience sake can make the use of water soluble immersion media acceptable with achromats but where detail is required, you cannot overlook the deleterious effects of refraction that most achromats maintain and only exacerbated by a lower N.A. and imperfect homogeneity. There is a reason that water immersion and glycerin immersion are not that common and those are also why confocal water immersion is done with apochromats mostly. 1.2 N.A. through an achromat is not the same as 1.2 N.A. through a fluorite or apochromat.

LouiseScot
Posts: 488
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Re: Glycerol Immersion Objectives

#10 Post by LouiseScot » Thu Apr 08, 2021 1:00 pm

apochronaut wrote:
Thu Apr 08, 2021 12:37 pm
As with so many things , there is theory and then there is empirici.
There does seem to be a bit of a phobia that plagues newly hatched microscopists when it comes to oil immersion. It is a little difficult to master but immersion is immersion and outside of an extra minute or two to clean up a bit of oil, the intricacies are pretty similar between the various immersion media but the results different. Oil has the advantage though, of being much more common, so the glass being utilized on a broad scale for preparation is more homogeneous with oil, more widely available and cheaper.
Between the categories of objective colour correction , stepping around the immersion debate, the results speak for themselves. If one is content to gaze at pretty colours and shapes, then working at lower N.A. for convenience sake can make the use of water soluble immersion media acceptable with achromats but where detail is required, you cannot overlook the deleterious effects of refraction that most achromats maintain and only exacerbated by a lower N.A. and imperfect homogeneity. There is a reason that water immersion and glycerin immersion are not that common and those are also why confocal water immersion is done with apochromats mostly. 1.2 N.A. through an achromat is not the same as 1.2 N.A. through a fluorite or apochromat.
Hi again

Of course, I have a standard 100x/1.25 oil immersion achromat and have had some quite good results from it and don't have a problem using it. I have the option of using monochromatic light sources and camera so CA is not a problem. Anyway, I've now purchased this 100x/1.30 glycerin immersion UV fluorite because it looked interesting! I should get it (from Japan) maybe Monday. I might struggle to get decent results from it though perhaps stopping the NA down will help. If I had to use it at 1.0, that would still be ok. Just using it at 0.85 means that I wouldn't gain anything over my 40x/0.85 fluorite. I'll start at the minimum collar setting of 0.8 and work my way up :) I'll just have to try it and see!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

apochronaut
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Re: Glycerol Immersion Objectives

#11 Post by apochronaut » Thu Apr 08, 2021 2:31 pm

Presumably your monochromatic condition is close to one of the wavelengths that the objective is corrected for?

LouiseScot
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Re: Glycerol Immersion Objectives

#12 Post by LouiseScot » Thu Apr 08, 2021 2:44 pm

apochronaut wrote:
Thu Apr 08, 2021 2:31 pm
Presumably your monochromatic condition is close to one of the wavelengths that the objective is corrected for?
Hiya

I can set up a source for a monochromatic narrow band within any major colour band. I currently have 415nm and 450nm source in the blue-violet and blue ranges. I could just as easily use a green LED. I don't know the specifics of the Nikon fluorite objective - I'll probably have to experiment. Obviously, as it's an epifluorescence objective it will transmit uv and the usual colours of fluophores. But I'm not intending to use it for that!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

LouiseScot
Posts: 488
Joined: Tue Jul 07, 2020 1:51 pm
Location: Scotland

Re: Glycerol Immersion Objectives

#13 Post by LouiseScot » Fri Apr 09, 2021 3:01 pm

I took delivery of the objective a little while ago! Have ordered some borosilicate 33 coverslips (n=~1.47-1.48) so will see how I get on with them in combination. Whilst I wait for the boro coverslips I'll try the objective with ordinary glass ones and see what happens!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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