Diatom microscopy using 254nm light

Images made through a microscope. All subject types.

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jmc
Posts: 238
Joined: Fri Apr 17, 2020 3:14 am

Diatom microscopy using 254nm light

Post by jmc »

As some of you know, I've been working on a UV transmission microscope to help with my research into sunscreens and skin cancer prevention. I've also been using it to look at some diatoms using 365nm and 313nm light. When I built it though, I wanted to make sure it would be usable at shorter wavelengths if I ever wanted to try that out. Recently I have been trying to see what can be done using 254nm light, just to see if I could get anything, and I have some results to share.

This was a simple brightfield image at 254nm, using my quartz based diatom slide. The camera is a monochrome converted Nikon d850 with a fused silica sensor coverglass window. Light was a 254nm 3W germicidal low pressure mercury bulb, and I filtered the light going to the camera with the combination of a 254nm forenics imaging filter, and a Semrock 260/16 brightline filter. The objective was a 10x Zeiss Ultrafluar NA 0.2. Single image only (no stacking) shown as the full field of view, and cropped to show the central diatom (both of these have been reduced in resolution for sharing) and then a very tight crop shown at the original pixel resolution of the image.
DSC_5690 Sem below Sirchie ab full lab small.jpg
DSC_5690 Sem below Sirchie ab lab small.jpg
DSC_5690 Sem below Sirchie cropped lab.jpg
This is very much a 'work in progress' and working at such a short wavelength presents major challenges (issues with camera sensitivity for one), and is a big safety challenge as 254nm can be very damaging to eyes and skin, but wanted to share in case it was of interest.
Jonathan Crowther

Vincent Zhang-1968
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Joined: Sun Feb 13, 2022 6:17 pm
Location: Hebei province,China

Re: Diatom microscopy using 254nm light

Post by Vincent Zhang-1968 »

Thanks for sharing. I think the details are quite good for a NA 0.2 objective lens.

jmc
Posts: 238
Joined: Fri Apr 17, 2020 3:14 am

Re: Diatom microscopy using 254nm light

Post by jmc »

Vincent Zhang-1968 wrote:
Tue Jul 19, 2022 1:16 am
Thanks for sharing. I think the details are quite good for a NA 0.2 objective lens.
No problem. Yes, it is pretty good for an NA 0.2 objective. I'm struggling with some aspects of the process though. The camera has very little sensitivity that far down into the UV (even though it is a monochrome conversion with the microlenses and Bayer array removed, a BSI sensor, and a fused silica coverglass). The image above was 30s exposure at ISO 1000. I cannot get any form of live view to help with focusing due to the low sensitivity. Even with the Zeiss Ultrafluar, when it is in focus in visible light it is way out of focus at 254nm. So there is a lot of trial and error with positioning. I can imagine with higher magnification objectives, this will become even more of a challenge.
Jonathan Crowther

rjlittlefield
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Re: Diatom microscopy using 254nm light

Post by rjlittlefield »

jmc wrote:
Wed Jul 20, 2022 12:46 am
when it is in focus in visible light it is way out of focus at 254nm. So there is a lot of trial and error with positioning.
Have you tried measuring the difference in focus distances and correcting by that fixed amount when switching from visible to 245nm? I expect the method will fail, but I am curious to know how.

--Rik

jmc
Posts: 238
Joined: Fri Apr 17, 2020 3:14 am

Re: Diatom microscopy using 254nm light

Post by jmc »

rjlittlefield wrote:
Wed Jul 20, 2022 9:27 am
jmc wrote:
Wed Jul 20, 2022 12:46 am
when it is in focus in visible light it is way out of focus at 254nm. So there is a lot of trial and error with positioning.
Have you tried measuring the difference in focus distances and correcting by that fixed amount when switching from visible to 245nm? I expect the method will fail, but I am curious to know how.

--Rik
Yes, that would be the plan long term. As I'd only be likely to use a couple of objectives at 254nm, hopefully I'll be able set of some sort of correction table.
Jonathan Crowther

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