Diatom imaged with 313nm light

Images made through a microscope. All subject types.

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Re: Diatom imaged with 313nm light

Post by rjlittlefield »

rjlittlefield wrote:
Thu Jun 02, 2022 10:11 am
jmc wrote:
Thu Jun 02, 2022 12:53 am
If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used.
Another possible factor: at same NA, the light cones ** *** ******* will be narrower, in proportion to the RI of the immersion fluid.

So where NA 0.95 requires light rays that are angled at 71.8 degrees off-axis, NA 1.0 in RI 1.51 requires only 41.5 degrees off axis.
In earlier post I had written "at the subject", thinking in a moment of craziness that we were dealing with diatoms that were dry versus immersed.

But even with subjects mounted under coverslips, those angles apply for light between the coverslip and the objective.

Intuitively, it seems like this should make the immersion objective simpler to design and manufacture, since the dry objective has to bend the light more while maintaining the same accuracy in optical path length for all rays.

--Rik

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Re: Diatom imaged with 313nm light

Post by Macro_Cosmos »

jmc wrote:
Thu Jun 02, 2022 12:53 am
Your findings make sense to me. If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used. Out of interest, was whether or not the condenser was used with immersion fluid looked at as part of the test?
Here is what I did:
- dry top lens (U-TLD), dry objective (even immersion ones, did not use immersion fluid)
- dry top lens, correct immersion
- oil top lens (U-TLO), oiled but not in contact with the slide, as my stage's well is not deep enough... objectives all dry
- oil top lens, same as above but with correct immersion
- oil top lens, correctly adhered to the slide and correction objective immersion
I also did this for reflected light.

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Re: Diatom imaged with 313nm light

Post by jmc »

Macro_Cosmos wrote:
Fri Jun 03, 2022 9:46 am
jmc wrote:
Thu Jun 02, 2022 12:53 am
Your findings make sense to me. If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used. Out of interest, was whether or not the condenser was used with immersion fluid looked at as part of the test?
Here is what I did:
- dry top lens (U-TLD), dry objective (even immersion ones, did not use immersion fluid)
- dry top lens, correct immersion
- oil top lens (U-TLO), oiled but not in contact with the slide, as my stage's well is not deep enough... objectives all dry
- oil top lens, same as above but with correct immersion
- oil top lens, correctly adhered to the slide and correction objective immersion
I also did this for reflected light.
Thanks.
Jonathan Crowther

jmc
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Re: Diatom imaged with 313nm light

Post by jmc »

Been preparing a few images for a talk I'm doing later this year on UV imaging, so thought I would share some here. These were taken using bright field at 313nm with a Leitz 40x NA 0.65 objective and a Zeiss quartz condenser (NA 0.85). Objective and condenser were used with glycerine immersion. Camera was a monochrome converted Nikon d800. Single images only (no stacking). Originally they were between 4000 and 7000 pixels across, so I have reduced the sizes for sharing here.
DSC_9836.JPG
DSC_9830.JPG
DSC_9825 313nm.JPG
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Re: Diatom imaged with 313nm light

Post by jmc »

Was playing around today with a rather unusual objective - a 170x Leitz Q NA 0.5 reflecting objective, designed for 400mm tube length microscopes. It was originally built for UV and visible imaging from 220nm to 700nm. Even though it was absolutely not designed for use with a 160mm tube length system, and should be used with its own reflecting condenser, I decided to give it a go anyway and got the following at 313nm. Single image and no stacking.
DSC_9972 365nm small.jpg
This was part of a diatom I have imaged before in bright field at 313nm (the 2nd image in my previous post). Even though I setup the system for bright field, I got a darkfield image. I had closed the iris of the condenser right down, so I presume a very small spot was illuminating the sample. As the objective is a mirror lens, it has a small central mirror, and I presume (again) that this mirror blocked the directly transmitted light, only letting scattered light through, hence producing a darkfield image.

Resolution is a bit lacking and depth of field very shallow, but then it was a low NA objective (0.5) and was being used well out of the design specs it was designed for. The working distance was huge for the magnification at about 3mm, but I expected it to be large as it is a mirror lens.

It's not something I'd use all the time, but thought UVB darkfield microscopy was pretty cool, hence I am sharing.

Here's what the objective looks like.
20220628_224945small.jpg
20220628_224955small.jpg
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Re: Diatom imaged with 313nm light

Post by Vincent Zhang-1968 »

Thanks for sharing these useful information! I wonder if there is a big difference between nikon UV-F 100X/1.30 and 100X/1.30 Fluor objective lens in UV light tranmission?
Attachments
UV-F.jpg

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Re: Diatom imaged with 313nm light

Post by jmc »

Vincent Zhang-1968 wrote:
Thu Jun 30, 2022 6:36 pm
Thanks for sharing these useful information! I wonder if there is a big difference between nikon UV-F 100X/1.30 and 100X/1.30 Fluor objective lens in UV light tranmission?
No problem. While I have not tested the 100x Fluor, I suspect it will be similar in terms of transmission to the UV-F 100x. However, transmission is only part of the story. These objectives weren't designed for imaging in the UV, but for transmitting light to allow for fluorescence, and then imaging in the visible light. If you want to use it for imaging at, for instance 365nm, then keep in mind that they may not be corrected for imaging in the UV, and may not be as sharp as they are in the visible region.
Jonathan Crowther

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Re: Diatom imaged with 313nm light

Post by jmc »

Quick follow up to my previous UVB diatom images. I went back to the slide with a 100x Leitz UV NA 1.2 glycerine immersion objective and got the following at 313nm. Single image, no stacking. One image which was almost the full frame captured and then a crop from original showing part of the top right of the image (both have been reduced in resolution for sharing).
DSC_9858mod vsmall.jpg
DSC_9858mod cropped tr vsmall.jpg
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Re: Diatom imaged with 313nm light

Post by Vincent Zhang-1968 »

jmc wrote:
Fri Jul 01, 2022 1:30 am
Vincent Zhang-1968 wrote:
Thu Jun 30, 2022 6:36 pm
Thanks for sharing these useful information! I wonder if there is a big difference between nikon UV-F 100X/1.30 and 100X/1.30 Fluor objective lens in UV light tranmission?
No problem. While I have not tested the 100x Fluor, I suspect it will be similar in terms of transmission to the UV-F 100x. However, transmission is only part of the story. These objectives weren't designed for imaging in the UV, but for transmitting light to allow for fluorescence, and then imaging in the visible light. If you want to use it for imaging at, for instance 365nm, then keep in mind that they may not be corrected for imaging in the UV, and may not be as sharp as they are in the visible region.
Thanks for your reply!

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Re: Diatom imaged with 313nm light

Post by Vincent Zhang-1968 »

During UV photoraphy, I also meet this phenomenon on the edge of diatoms, I think it's artifact, but don't know how to improve it, focus stacking does not work.
Attachments
artifact.jpg

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Re: Diatom imaged with 313nm light

Post by jmc »

Vincent Zhang-1968 wrote:
Sun Jul 03, 2022 6:44 pm
During UV photoraphy, I also meet this phenomenon on the edge of diatoms, I think it's artifact, but don't know how to improve it, focus stacking does not work.
I've struggled with it too, which is one of the reasons I tend not to do stacking with these images.
Jonathan Crowther

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Re: Diatom imaged with 313nm light

Post by LouiseScot »

jmc wrote:
Mon Jul 04, 2022 12:39 am
Vincent Zhang-1968 wrote:
Sun Jul 03, 2022 6:44 pm
During UV photoraphy, I also meet this phenomenon on the edge of diatoms, I think it's artifact, but don't know how to improve it, focus stacking does not work.
I've struggled with it too, which is one of the reasons I tend not to do stacking with these images.
I've seen it too - to do with the thickness of the diatom? Only occurs with some.
Louise

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Re: Diatom imaged with 313nm light

Post by jmc »

LouiseScot wrote:
Tue Aug 16, 2022 3:33 pm
jmc wrote:
Mon Jul 04, 2022 12:39 am
Vincent Zhang-1968 wrote:
Sun Jul 03, 2022 6:44 pm
During UV photoraphy, I also meet this phenomenon on the edge of diatoms, I think it's artifact, but don't know how to improve it, focus stacking does not work.
I've struggled with it too, which is one of the reasons I tend not to do stacking with these images.
I've seen it too - to do with the thickness of the diatom? Only occurs with some.
Louise
I'm not sure why it happens, but looking at my image above where it is obvious, it seems to occur at the edge where the diatom is starting to curve away from the coverslip. So perhaps this small gap is the reason. As I say though, I am not sure, and I've not looked at enough samples to get a better feel for where is does and doesn't happen.
Jonathan Crowther

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Re: Diatom imaged with 313nm light

Post by LouiseScot »

jmc wrote:
Wed Aug 17, 2022 12:56 am
LouiseScot wrote:
Tue Aug 16, 2022 3:33 pm
jmc wrote:
Mon Jul 04, 2022 12:39 am

I've struggled with it too, which is one of the reasons I tend not to do stacking with these images.
I've seen it too - to do with the thickness of the diatom? Only occurs with some.
Louise
I'm not sure why it happens, but looking at my image above where it is obvious, it seems to occur at the edge where the diatom is starting to curve away from the coverslip. So perhaps this small gap is the reason. As I say though, I am not sure, and I've not looked at enough samples to get a better feel for where is does and doesn't happen.
I brought it up on the other forum over a year ago when I was trying out a just purchased Nikon F100-UV glycerine immersion lens https://www.microbehunter.com/microscop ... 60#p102284 Was imaging with a mono light source (415nm) and mono camera.

Louise

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Re: Diatom imaged with 313nm light

Post by Smokedaddy »

Care to share a few postings of your finalized microscope setup?

-JW:

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