Diatom imaged with 313nm light

Images made through a microscope. All subject types.

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jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Minor update. I got a Newport Highres-2 USAF test slide to help with my work - https://www.newport.com/f/high-resolution-test-targets

The one I have is a positive one, with opaque lines on a clear quartz slide. I've done some resolution testing at different wavelengths with that Leitz 100x NA 1.2 UV objective (the condenser is a quartz Zeiss one NA 0.85). Both objective and condenser were use with glycerine immersion fluid, and there was a 0.35mm quartz coverslip (again with a small amount of glycerine between the coverslip and slide.

The slide is divided into lines of different thickness defined by Group (4 to 11) and Element (1 to 6) as outlined in the link above. Group 11 is the smallest, and the lines get smaller from Element 1 to 6 (in Group 11, it goes 1, 2, 3 and then 3 circles instead of the numbers, presumably as they are too small).

Here's the images of the test slide imaged at different wavelengths.

Firstly, 313nm.
DSC_9412 313nm mono2dng lab small.jpg
Then 365nm.
DSC_9399 365nm mono2dng lab small.jpg
And finally 546nm.
DSC_9394 visible mono2dng lab small.jpg
Using 313nm light it looks to be able to resolve down to about 150nm, which is roughly in line with what the maths says.
Jonathan Crowther

René
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Re: Diatom imaged with 313nm light

Post by René »

Well done! Quite dramatic to see the resolution improving.
BTW, are you able to make an image with a normal 100x oil immersion objective? I expect that to give a better image than such a specialized UV objective in the normal light region.

Best wishes, René

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

René wrote:
Thu Apr 28, 2022 2:45 am
Well done! Quite dramatic to see the resolution improving.
BTW, are you able to make an image with a normal 100x oil immersion objective? I expect that to give a better image than such a specialized UV objective in the normal light region.

Best wishes, René
Thanks René, yes it was really nice to see the change. In theory that should be doable, although at the moment I do not have a suitable 100x high NA objective to try it with. I'd also need to change the condenser (currently using a Zeiss quartz NA 0.85 one) and photoeyepiece (currently a Lomo quartz one) to try and show the best case scenario with with the normal objective.

The Leitz UV objectives are interesting. I've not be able to find out much about them at all, but I suspect they were made to be competitive against the Zeiss Ultrafluars, and to be usable in the UV and Visible region. Are they as good as an Olympus Splan APO 100x in the visible region, probably not, however I have been very surprised by them and how sharp they are at different wavelengths.

Jonathan
Jonathan Crowther

Lou Jost
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Re: Diatom imaged with 313nm light

Post by Lou Jost »

I have the Nikon version of this (160mm tube length "UV-F" 100x 1.20) but have never tried it. This result makes me want to try!

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Lou Jost wrote:
Thu Apr 28, 2022 12:05 pm
I have the Nikon version of this (160mm tube length "UV-F" 100x 1.20) but have never tried it. This result makes me want to try!
Hi Lou, I believe (though I admit, I have never tested) that the Nikon UV-F 100x will have different UV transmission to the Leitz UV I was using. Let me explain.

The Leitz UV 100x I used has no glass in it. It is similar to the Zeiss Ultrafluars (which is quartz and calcium fluoride lenses) and was designed to be be used for imaging down into the deep UV (<300nm). The Ultrafluars were designed to show minimal focus shift with wavelength even down below 300nm vs visible light, and the Leitz UV behaves like that when I try it at 546nm, 365nm and 313nm. If I measure the transmission of these types of lenses (Leitz UV and Zeiss Ultrafluar) I get a pretty flat line between 280nm and 420nm. For example, a Leitz 16x UV;
Leitz 16x UV objective.jpg
I am assuming now that the Nikon UV-F is designed for fluorescence imaging. It will have good transmission in the UV (mainly the longer wavelength UVA range) but will be designed for imaging in the visible region. It will have glass lens elements. I don't have any Nikon UV-F lenses to test, although they are on my list to track down, but here is the transmission spectrum through an Olympus UVFL 10x which is one of their fluorescence objectives;
Olympus 10x UVFL.jpg
The Olympus 10x UVFL transmission drops towards the short wavelength end, although still has pretty good transmission at 365nm. How good it is at imaging there I don't know as I've never tried it, but it will let the light through. By the time you get to 313nm though, it is essentially blocking all the light.

As I say though I have not tested any of the Nikon UV-F objectives, so this is currently speculation on my behalf based on on what I have seen with the Olympus fluorescence objectives.

I've found the 'UV' labeling of microscope parts really confusing to follow during my research. From what I can tell, usually when a microscope says UV it means 'UV fluorescence' and that typically means UVA (the 365nm line).
Jonathan Crowther

Lou Jost
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Re: Diatom imaged with 313nm light

Post by Lou Jost »

Thanks, I think you are right about the Nikon UV-F series being aimed at fluorescence rather than deep UV. Too bad.

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Lou Jost wrote:
Fri Apr 29, 2022 8:35 am
Thanks, I think you are right about the Nikon UV-F series being aimed at fluorescence rather than deep UV. Too bad.
No problem Lou. If you have a suitable light source, camera and filters for imaging at 365nm then it is probably still worth a try at that wavelength. But I wouldn't bother if you need to buy stuff in for it.
Jonathan Crowther

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Lou Jost wrote:
Thu Apr 28, 2022 12:05 pm
I have the Nikon version of this (160mm tube length "UV-F" 100x 1.20) but have never tried it. This result makes me want to try!
Lou. I managed to find one of these UV-F 100x objectives for sale in Europe and have bought it. When it arrives, I'll do some testing on it and let you know how I get on. Jonathan
Jonathan Crowther

Lou Jost
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Re: Diatom imaged with 313nm light

Post by Lou Jost »

Great, I very much look forward to knowing the results! Thank you. I hope it at least has some useful properties, even without deep transmission.

Scarodactyl
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Re: Diatom imaged with 313nm light

Post by Scarodactyl »

Normal Nikon cf fluorescence-optimized objectives were marked Fluor (equivalent to today's S Fluors). I wonder how the UV F ones relate.

Lou Jost
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Re: Diatom imaged with 313nm light

Post by Lou Jost »

I just found this article which says the UV-F 100x only transmits down to 325-330nm

https://ecovis.org.au/wp-content/upload ... search.pdf

Perl
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Re: Diatom imaged with 313nm light

Post by Perl »

Nice Work Johnathan !
Best Regards
Pär
****** Seeing is Believing ******

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Thanks all.

Scarodactyl, in the Nikon CF objectives brochure the UV-F 100x is included with the Fluor ones in the Epi fluorescence section - Featuring extra-high light transmission into the ultraviolet excitation range the high NA fluor dry and fluor oil objectives are ideal for observation and photomicrography of low light fluorescence images. The newly developed fluor 40x and 100x oil immersion objectives are designed specifically for high ultraviolet transmission down to the 340nm range for fluorochromes like FURA-2. The glycerine immersion CF UV-F 100x may be recommended where a water soluble immersion medium is required.

I'm not familiar with Nikons range of objectives or the dates they were made, but do the UV-F ones predate the Fluor ones? I have seen other glycerine immersion UV-F ones with different magnifications but they are not mentioned here at all.

Funnily enough at the same time that I bought the 100x UV-F I also got a 10x Fluor NA 0.5, so will measure the transmission of that one as well.
Jonathan Crowther

Lou Jost
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Re: Diatom imaged with 313nm light

Post by Lou Jost »

Jonathan, there is a whole series of UV-F objectives (10x, 20x, 40x, 100x), all glycerine immersion objectives. Fluor objectives have always been around and continue to be made, and are completely independent of this UV-F line.

The 40x UV-F has an NA of 1.30, one of the highest NAs for such a low power objective.
Last edited by Lou Jost on Wed May 04, 2022 7:23 am, edited 1 time in total.

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Lou Jost wrote:
Wed May 04, 2022 5:46 am
Jonathan, there is a whole series of UV-F objectives (10x, 20x, 40x, 100x), all glycerine immersion objectives. Fluor objectives have always been around and continue to be made, and are completely independemt of this UV-F line.

The 40x UV-F has an NA of 1.30, one of the highest NAs for such a low power objective.
Ah right, thanks Lou.
Jonathan Crowther

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