Visible, UVA and UVB transmission microscopy of a diatom

[Andy Perrin]

Woooow! That is a very impressive setup. Why does this diatom look so different from the previous photos? It’s not oval shaped etc. 

[JMC]

37 minutes ago, Andy Perrin said:

Woooow! That is a very impressive setup. Why does this diatom look so different from the previous photos? It’s not oval shaped etc. 

The chap made me a slide with about 20 different diatoms on it. I'm having a look around it, so am imaging different ones.

[Andrea B.]

Jonathan, you are having one of the best UV adventures I've ever seen! Thank you for sharing all this good stuff with UVP.

[JMC]

19 hours ago, Andrea B. said:

Jonathan, you are having one of the best UV adventures I've ever seen! Thank you for sharing all this good stuff with UVP.

Thanks Andrea. It has been fascinating looking at these tiny samples. I'm reminded of William Blake - To see a world in a grain of sand.

 

Last night I tried using a Leitz 100x NA 1.2 objective at 313nm to look at one. I'm still processing the image but will share when I get the chance.

[JMC]

Here's the latest image taken at 313nm on my microscope. A different diatom again, and this time done with a Leitz 100x NA 1.2 UV objective. Only shows the central part of it due to the extreme magnification. I used glycerine immersion between the condenser and the slide, and between the coverslip and objective. About 12 images stacked in Zerene and then with a bit of post processing (the regular pattern across the surface gave me some issues with stacking). Reduced it in resolution for sharing, as the original is full frame from my Nikon d800.

 

Going in closer to the original image gave the following.

 

The 'round' features are about 500nm across and the gaps between them about 200nm.

 

Here's what the whole thing looks like, taken using a 32x Ultrafluar objective.

 

And finally the objective itself.

[colinbm]

Amazing Jonathan

[JMC]

On 3/13/2022 at 9:53 AM, colinbm said:

Amazing Jonathan

Thanks Colin. Been fascinating looking at these sample like this.

[colinbm]

So would I, but it wont happen unfortunately.
I gather these are the cells that we can see ?

[JMC]

3 minutes ago, colinbm said:

So would I, but it wont happen unfortunately.
I gather these are the cells that we can see ?

The diatoms are the silica structures produced by certain algae as far as I know. These are what gets left behind when the algae dies.

[JMC]

New update to the work. I bought a Newport Highres-2 microscope resolution test slide, so I can put some numbers to what I am seeing - https://www.newport.com/f/high-resolution-test-targets

 

This is a USAF slide with opaque lines on a quartz slide (so is UV transparent). It also so has some very small features on it - down to 137nm lines. It arrived yesterday so I got a photo of it using the Leitz 100x NA 1.2 UV objective, which I used for the image in my post from the 13th March. Imaging done with my monochrome d800 from MaxMax.

 

This was the 313nm image (reduced in resolution by about a factor of 2 for sharing).

 

In Group11, Element 1 is 244nm lines, 2 is 218nm, 3 is 194nm, 4 (the top 'dot) is 173nm, 5 (the middle 'dot') is 154nm and 6 (the bottom 'dot) is 137nm. it certainly looks to be getting down towards 150nm in terms of resolution. Group 11, Element 6 is the smallest feature on the slide.

 

Out of the interest, here is the 365nm one and visible light one (546nm) taken with the same setup.

 

 

Nice to be able to put some numbers to what I'm seeing, and not too shabby for a home modified microscope. Now if only I could get it to work at 254nm....

 

Here's the full slide taken with a 4x objective in visible light. Group 11 is buried somewhere in the middle.....

[Andy Perrin]

Cool! Did you try it with glycerin?

[Stefano]

You are clearly gaining resolution in UV. This shows that you are diffraction-limited.

[JMC]

21 minutes ago, Andy Perrin said:

Cool! Did you try it with glycerin?

Yes, from bottom to top it was antique zeiss quartz condenser (NA 0.85), glycerine, test slide, small amount of glycerine, 0.35mm quartz coverslip, glycerine, objective (Leitz 100x NA1.2 UV). Then after it has gone through a block of fused silica in the trinocular head (where a beamsplitter originally sat), it's then a Lomo quartz photoeyepiece, any filters, and then the camera.

[JMC]

Been making some images over the last few days for a talk i'm going to be giving later this year, and thought I would share some here. These have been reduced in resolution for sharing - typically the originals are 3000 - 7000 pixels across and these are 1000 on the longest axis.

 

Firstly the microscope slide itself with the diatom arrangement. This is a slide which was made for me using fused silica/quartz slide and coverslip so it can be used for UV. The diatoms were mounted with a small amount of gelatin. This image was just a visible light one with a 4x objective and using oblique illumination from below.

 

Now the fun ones. These are done at 313nm with a Zeiss quartz NA 0.85 objective and Leitz 40x NA 0.65 UV objective. At 313nm and saved as JPEGs in the camera my monochrome Nikon d800 does give a slight colour cast. I normally get rid of that and show them as just grey images, but I have kept it here - I quite like it, it reminds me of sepia.

 

Using 313nm gives a much improved resolution vs visible light and even 365nm. With this objective at 313nm, I am seeing features down to around 300nm based on what I can see in the images. Does make me wonder what imaging at around 250nm would do.

 

 

 

 

 

 

 

 

All the images above were single images - no stacking.

 

As a final image, the one below is a stack of two shots.

 

The two images which were stacked together were as follows;

 

 

Not had a lot of success with stacking diatom images, but these two were sufficiently far apart in terms of focus for it not to get confused.

[colinbm]

Truly amazing & so sharp, excellent Jonathan

 

[JMC]

8 hours ago, colinbm said:

Truly amazing & so sharp, excellent Jonathan

 

Cheers Colin.

[Andy Perrin]

Fantastic work, Jonathan!

[JMC]

3 hours ago, Andy Perrin said:

Fantastic work, Jonathan!

Thanks Andy.

[dabateman]

Very good. I look forward to seeing if you can push it to 250nm with a UVC light.

[JMC]

16 hours ago, dabateman said:

Very good. I look forward to seeing if you can push it to 250nm with a UVC light.

Cheers David. 250nm will be doable by the microscope, but at the moment I do not have the other hardware required for it (especially a suitable light).

[photoni]

Jonathan they are impressive "optical" images
give meaning to deep UV images

Bravo !

[JMC]

12 hours ago, photoni said:

Jonathan they are impressive "optical" images
give meaning to deep UV images

Bravo !

Thanks Toni.

[JMC]

A final update for now (as I really must get some work done). Going from the cm scale down to the nm scale on the microscope with a diatom and a 100x objective and 313nm light - https://jmcscientificconsulting.com/uv-microscopy-its-a-matter-of-perspective/

[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.

 

As a comparison, here's the same diatom (different magnification) also at 313nm.  The area I just imaged is at the upper right of the one below.

 

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, 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.

 

[dabateman]

Well that is quiet an interesting image.

That objective must also be supper rare. I think it might be made for a reflectance microscope,  as the working distance is quite large at 2.5mm.

Thats a huge working distance for that magnification,  most likely designed to look at rock or concrete samples. 

The image looks quite nice and if you did hit a dark field sweet spot, than you should expect up to 10x gain in resolution.