Just got my diatom test slide!

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josmann
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Just got my diatom test slide!

#1 Post by josmann » Tue Nov 23, 2021 12:10 am

This thing is sick!

I'm doing holiday stuff so I can't play with it too much but wow this is really a neat product. This image is a quick picolay focus stack (maybe 8 images?) with an Olympus 20x SPlanApo lens on a BH2. One really cool thing to see was when I switched to my 40x SPlan which has the same NA (.70) you truly don't see much change at all other than the extra zoom. I tried out the 40x SPlanApo as well (.95 NA) which definitely sharpens up the pores on the left diatom and just starts to reveal some criss-cross patterns in the middle diatom but I think I'll need to try the 100x to really make that one pop.
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Re: Just got my diatom test slide!

#2 Post by dtsh » Tue Nov 23, 2021 1:28 am

I suspect we have slides from the same source.
viewtopic.php?p=112212#p112212

You've done a much better job at imaging it than I have.

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Re: Just got my diatom test slide!

#3 Post by KurtM » Tue Nov 23, 2021 2:11 am

So where are these test slides coming from? Diatom Lab?
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Re: Just got my diatom test slide!

#4 Post by josmann » Tue Nov 23, 2021 2:21 am

Should've mentioned - this came from www.diatomshop.com

They've got a lot of other diatoms and calibration stuff as well. Probably will be making another order with them some day - I have an interest in optical characterization and calibration.
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Re: Just got my diatom test slide!

#5 Post by josmann » Tue Nov 23, 2021 5:30 am

dtsh wrote:
Tue Nov 23, 2021 1:28 am
You've done a much better job at imaging it than I have.
Haha, thanks but that 40x plan photo looks pretty darn close by my judgment! You're lacking just a little bit of numerical aperture in the objective but it's close.

I have a 1.40 NA SPlanApo 100x that I haven't tested out yet due to lack of quality oil. Really excited to see how that turns out!
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Re: Just got my diatom test slide!

#6 Post by Phill Brown » Tue Nov 23, 2021 5:51 pm

And then there is dark field and strew slides.
Antique slides are often excellent value and quality.
Good luck with capping the spending.

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Re: Just got my diatom test slide!

#7 Post by josmann » Tue Nov 23, 2021 8:05 pm

Phill Brown wrote:
Tue Nov 23, 2021 5:51 pm
Good luck with capping the spending.
Oh don't worry, I think my scope/video hardware spend will keep the slide purchasing in check ;)
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Re: Just got my diatom test slide!

#8 Post by josmann » Fri Dec 03, 2021 8:32 pm

Okay, so I beat level 4 (Navicula oblonga) using two techniques: 100x SPlanApo with double immersion AAC condenser and same objective with Amscope oil immersion darkfield condenser. Still haven't been able to resolve the poroids in the 5th diatom (tightly spaced, separation around 110nm). It's recommended by Diatom Lab to use polarized light to bring these out so I'll have to rig something up for that.

The striae of this frustule are composed of lineolae running transverse to the direction of each stria. According to diatomshop, the spacing between these lineolae averages 140nm. I think I'm most satisfied to get them resolved in BF! I do have to pull a lot more contrast out in post for brightfield, whereas they are more clear natively in DF.
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Re: Just got my diatom test slide!

#9 Post by Hobbyst46 » Fri Dec 03, 2021 8:40 pm

140nm resolution in BF... :shock: :? :o

From the Diatoms of North America site:
"Areolae number 32-40 in 10 µm and are distinguishable in LM with critical illumination", so they are spaced 0.25-0.33
micrometer apart.

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Re: Just got my diatom test slide!

#10 Post by josmann » Fri Dec 03, 2021 9:30 pm

Hobbyst46 wrote:
Fri Dec 03, 2021 8:40 pm
140nm resolution in BF... :shock: :? :o

From the Diatoms of North America site:
"Areolae number 32-40 in 10 µm and are distinguishable in LM with critical illumination", so they are spaced 0.32 micrometer apart.
Hi Hobbyst,

I believe these Diatoms are carefully selected by diatomshop to conform to the parameters they specify. Here is the associated card for their variant of this species: http://www.diatomlab.com/gallery/Diatom ... blonga.jpg

And some more info on the specimens: http://www.diatomlab.com/diatom-test-sl ... n-2.0.html

They use a n=1.7 mountant which allows for a bit of super-resolution imaging - light which would normally diffract into uncapturable orders will propagate at less oblique angles in this medium allowing them to enter the objective and contribute to interference at the image sensor.

Right now I'm working on the P. nobilis using polarized light (with cheap plastic polarizing film). I'm juuuuust barely able to get something, although it's hard to tell if it isn't aliased false images.
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Re: Just got my diatom test slide!

#11 Post by Hobbyst46 » Fri Dec 03, 2021 10:38 pm

Thanks Jossman,

So Diatom-lab expect "super resolution" under the following conditions:

1. Their n=1.7 mountant - well, AFAIK the theoretical expressions for BF resolution (Abbe, Rayleigh) do not take into account the mountant layer. Anyway, sadly, Diatom Lab do not specify what it is or even sell it...

2. Their suggested illumination modes are not really BF. If the illumination you used was not plain Vanilla BF,
that explains the very nicely resolved image of N. Oblonga you posted.

Will be interesting to see the 0.11 micrometer of P. Nobilis !

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Re: Just got my diatom test slide!

#12 Post by josmann » Fri Dec 03, 2021 11:24 pm

Hobbyst46 wrote:
Fri Dec 03, 2021 10:38 pm
AFAIK the theoretical expressions for BF resolution (Abbe, Rayleigh) do not take into account the mountant layer
Yeah but there was still considerable development of microscopy theory subsequent to them! There are all sorts of edge cases which allow you to push and pull that limit around. Abbe's key insight, imo, was not the resolution limit equation, but rather the crucial insight that successful imaging of small objects requires capturing diffraction orders. That's the true limit to resolution - can you get all those orders together in the same location on your image sensor?
Hobbyst46 wrote:
Fri Dec 03, 2021 10:38 pm
2. Their suggested illumination modes are not really BF. If the illumination you used was not plain Vanilla BF,
that explains the very nicely resolved image of N. Oblonga you posted.
Nope - that one was indeed plain vanilla BF. Aplanat-achromat condenser oil immersed and Koehler aligned and set to 1.4 to match the objective. Second image is plain vanilla darkfield as well. I don't think they're saying you can't use BF - just that it'll be clearer with other methods.

For P. Nobilis, I did indeed need to go to polarization to resolve with any fidelity. I put a plastic film polarizer over the field lens and another underneath the dovetail of the trinoc head. Manually manipulated for extinction. White light was fairly rough, but the structures are definitely there. It was when I added a few blue and purple photo gels beneath the field lens polarizer that I was able to generate sharper images with more contrast. I was also doing exposures on the order of 10s for these.

Not quite as crisp as what diatomshop shows in their light image, but they've probably got a bit better hardware and a lot more experience :lol:: http://www.diatomlab.com/gallery/Diatom ... obilis.jpg
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Re: Just got my diatom test slide!

#13 Post by josmann » Fri Dec 03, 2021 11:57 pm

And here is a Gdrive folder with lossless full size PNGs if anyone wants to pixel peep:

https://drive.google.com/drive/u/0/fold ... ZCcUS1epOK
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Re: Just got my diatom test slide!

#14 Post by MichaelG. » Sat Dec 04, 2021 12:03 am

josmann wrote:
Fri Dec 03, 2021 11:24 pm

[…]

Not quite as crisp as what diatomshop shows in their light image, but they've probably got a bit better hardware and a lot more experience :lol:: http://www.diatomlab.com/gallery/Diatom ... obilis.jpg
Jason
Very impressive result, Jason

What does surprise me, however, is the general fuzziness of the SEM image that is provided for comparison
… and I also find it difficult to correlate the LM and SEM images on that page
… is it just me ?

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Re: Just got my diatom test slide!

#15 Post by josmann » Sat Dec 04, 2021 1:17 am

MichaelG. wrote:
Sat Dec 04, 2021 12:03 am
Very impressive result, Jason

What does surprise me, however, is the general fuzziness of the SEM image that is provided for comparison
… and I also find it difficult to correlate the LM and SEM images on that page
… is it just me ?

MichaelG.
Thanks Michael.

Refer to the photo here: https://diatoms.org/genera/pinnularia/guide

I believe what’s going on is the diatom frustule has inner and outer features. If you look at the low mag in both mine and that website’s photos, you’ll see a sort of small boxy striation in the larger stria. That is a window from the inner surface to the outer surface with the poroids (or vice versa - not sure). The poroids extend beyond this window, though. The glassy structures will be opaque to SEM which makes the nuance to the structure apparent whereas obviously it’s clear under LM so you don’t perceive the little window bit.

I hope that’s clear as mud - otherwise just furrow your brow at those pictures long enough until you get it.

I think the bottom right photo better shows what I’m seeing in the LM
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Re: Just got my diatom test slide!

#16 Post by MichaelG. » Sat Dec 04, 2021 1:40 am

Many thanks for the link
… It’s well past bedtime here, so I will furrow my brow in the morning when fully caffeinated !

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Re: Just got my diatom test slide!

#17 Post by Hobbyst46 » Sat Dec 04, 2021 9:00 am

Thanks for the very impressive and informative images !

I still wonder about the significance of the RI of the mounting medium in this case - except that the slide was probably very well prepared. The mounting layer is probably less than 0.1mm thick.

Also, I wonder how the 110-140nm resolution is specific to this one or these two diatoms. Diatoms are not "simple" targets. They scatter, refract, reflect, disperse, behave as selective lenses...

Combined blue-violet light and maximum extinction require extremely bright illumination. My previous experience with such setups was eye stress, and difficulty in focusing. All to make your posted images more impressive - and motivating.

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Re: Just got my diatom test slide!

#18 Post by Francisco » Sat Dec 04, 2021 12:41 pm

Very nice.

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Re: Just got my diatom test slide!

#19 Post by josmann » Sun Dec 05, 2021 3:13 am

I went back at it today, this time with cross-polarized darkfield illumination. However, I kept my objective's iris fully open to maximize NA.

The lineolae on N. oblonga are now very highly contrasted. They are perceptually white/black when optimally illuminated. I have provided a detail view of a deformed stria. I feel like this is worth noting as it shows there is not a continuation of the grating pattern indicating some sort of false image.

For P. nobilis I attempted a similar approach. The exposures for these subjects are on the order of 10s, so the camera display is extremely noisy. Much of my approach yesterday could be described as trial and error focusing. Today I used the eyepieces more and found some features which I could tell were absolutely definitely real. I saw a constellation of larger pores - two spaced diagonally somewhat far apart and a line of three others nearby. I used this as a reference point when examining the images in post so that, if I saw them, I knew I was actually focused on something. In the zoomed out photo, this constellation is about three quarters over and three quarters down. Refer to the detail image to find it. I know with certainty that those are real features - and I believe the nearby intensity modulations are smaller pores. The checkerboard modulations on the left side striae may be interference artifacts. I have uploaded lossless quality images to the Gdrive: https://drive.google.com/drive/u/0/fold ... ZCcUS1epOK

I also did some reading up on using high numerical aperture materials to elicit super-resolution. One technique developed in that last couple decades is using microspheres of high index material in contact with the subject. These couple evanescent EM waves which would normally only exist near the surface of the subject and cause them to propagate as light. Most papers I saw using this method stated a maximum resolution of down to 100-80nm which would agree well with diatom lab's stated resolutions. I wonder if their proprietary mountant is an adhesive (NOA170?) mixed with colloidal high RI particles? Or do you even need them if you're already using a high NA mountant? I don't imagine they're inclined to be forthcoming about their trade secret :)
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Re: Just got my diatom test slide!

#20 Post by Hobbyst46 » Sun Dec 05, 2021 7:26 am

1. Excellent images ! very exciting to see.

2. It was clear from the beginning (I think) that the highly resolved pattern was not an artifact. It is neither radial all over the image, nor parallel all over the image, or the like.

3. If recorded with a high NA objective, and the iris was fully open (the objective iris, not the condenser iris), then it was more COL than DF, IMHO. COL is supposed to improve resolution AFAIK.

4. The idea of mountant micro-spheres sounds tempting. It would probably require tailoring the RI and size of those micro/nano lenses. If so, certainly Diatom Lab will never tell what it is. NOA adhesives are supposedly based on somewhat similar novel components. I once considered trying NOA adhesives that boast NAs of 1.7+ but they were described as non-hardening. Besides, I worked with NOA 61 and found out that it does not fill frustules well: several types of diatoms contained air bubbles. So abandoned the NOA. In addition, NOA are expensives and shelf-time might be short.

5. I wonder how much of a challenge to view and focus under crossed polars plus DF.

For the exposure of 10s - what was the ASA setting ?
Last edited by Hobbyst46 on Sun Dec 05, 2021 9:36 am, edited 1 time in total.

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Re: Just got my diatom test slide!

#21 Post by MichaelG. » Sun Dec 05, 2021 7:34 am

Very rigorous observation, and great results there, Jason

… I shall take pleasure in looking at your lossless images later today.

MichaelG.

.
Edit: __ Whilst searching for any details of the ‘Diatom Cubed’ mountant … I happened-across this PhD thesis from 2017
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Re: Just got my diatom test slide!

#22 Post by josmann » Mon Dec 06, 2021 10:57 pm

Hobbyst46 wrote:
Sun Dec 05, 2021 7:26 am
4. The idea of mountant micro-spheres sounds tempting. It would probably require tailoring the RI and size of those micro/nano lenses. If so, certainly Diatom Lab will never tell what it is. NOA adhesives are supposedly based on somewhat similar novel components. I once considered trying NOA adhesives that boast NAs of 1.7+ but they were described as non-hardening. Besides, I worked with NOA 61 and found out that it does not fill frustules well: several types of diatoms contained air bubbles. So abandoned the NOA. In addition, NOA are expensives and shelf-time might be short.

5. I wonder how much of a challenge to view and focus under crossed polars plus DF.

For the exposure of 10s - what was the ASA setting ?
I sent an email to diatom lab shaking the tree a bit but they're holding on tightly to their coconuts. They would not expound at all on the physical mechanism. They do claim that they are the only lab able to achieve such results. I don't know if that means there's more to the SR than the high index mountant or just that they're the only ones who can prepare it in such a way to maintain good clarity.

Check out this paper on using microspheres to resolve down to 50nm: https://arxiv.org/ftp/arxiv/papers/1006/1006.4037.pdf

Figure 4 shows what you get when you scatter the spheres broadly on top of a specimen with small features... Honestly it does not seem like there is much to it - just get something with pretty high RI close to the subject. "Solid immersion lenses" is another concept to read up on.

I will read up more on this and maybe put together a little review in the future. High index microspheres are available from numerous sources off the shelf. They're not cheap, but not prohibitively expensive for a dedicated hobbyist either: https://www.cospheric.com/BTGMSc_solid_ ... icrons.htm Honestly, it seems like a fairly straightforward home experiment once you have spheres. You just need a good subject. It appears a common target is the lines on blu-ray disks.

ISO setting was 320 - I did these through a binoc port with a Varimag. Images with naked eye were pretty faint - fine details sort of like starlight.
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Re: Just got my diatom test slide!

#23 Post by josmann » Thu Dec 16, 2021 10:36 am

Okay, new hardware, new attempt. This time DIC. DIC greatly improves the contrast of my "constellation" of larger pores. They're now much more clear. A lot of the plaid-like patterning is gone which makes me suspect there was some interference at play there - the COL light definitely has a bit of coherence to it based on some other testing. I have more confidence that the markings that I am seeing now are real. I don't think I'm capturing this as cleanly as I can (there's more vibration today thanks to the storm hitting Claifornia), but even with the eyepieces I still can't say I see the small pores with a great amount of fidelity. I think DIC isn't necessarily the best technique to bring out these ultra small details - phase contrast might be a better fit. Don't have a 100x Ph objective yet, though.

Also, on a less exciting note, it appears that my S. phoenicenteron frustule is breaking at the top :( I don't think I mishandled the slide... Not sure if this is common to see with mounted diatoms.

High quality PNGs in same folder.
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Re: Just got my diatom test slide!

#24 Post by Hobbyst46 » Thu Dec 16, 2021 11:00 am

Different illumination modalities create different images, not necessarily artifacts.
Phase contrast is supposed to yield slightly worse resolution than BF, yet better contrast.

About the broke diatom: I have often noticed fractured diatoms in my slides, under oil immersion, but have not seen anything mentioned about it in literature. Why ? never happened to others ?

I mount mostly in Pleurax and occasionaly in Naphrax. Large diatoms are often broke after a session under oil immersion, using the 63X1.4 or 100X1.3 phase contrast objectives. Presumably, the tiny strain induced by the objective through the oil layer did it - and perhaps, it has to do with the rigidity of the mounting resin itself. How does one know for sure that the resin has completely solidified ? although my protocols follow the known instructions for mounting in those resins.

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Re: Just got my diatom test slide!

#25 Post by 75RR » Thu Dec 16, 2021 2:25 pm

.
Those are nice images!

It certainly is a challenge (and fun) trying to tease out the smallest of details in some diatoms.
josmann wrote:
Thu Dec 16, 2021 10:36 am
I think DIC isn't necessarily the best technique to bring out these ultra small details - phase contrast might be a better fit.
While I believe UV gets the most out of diatoms, I have found combining COL with Pol (COL+Pol) works better than DIC - Phase is a poor second (make that third) to either of these.

http://www.mikroskopie-ph.de/UV-diatoms-2011.pdf

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Re: Just got my diatom test slide!

#26 Post by Chas » Sat Jan 01, 2022 1:32 pm

Just thinking about Diatom lab 'holding onto their coconuts': perhaps they were using some variant of a 'solid immersion objective'? :
https://www.biorxiv.org/content/10.1101/373647v1.full

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Re: Just got my diatom test slide!

#27 Post by Hobbyst46 » Sat Jan 01, 2022 1:58 pm

75RR wrote:
Thu Dec 16, 2021 2:25 pm
While I believe UV gets the most out of diatoms, I have found combining COL with Pol (COL+Pol) works better than DIC - Phase is a poor second (make that third) to either of these.
http://www.mikroskopie-ph.de/UV-diatoms-2011.pdf
Thanks 75RR for this link ! never too late to read a previously-missed article !

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Re: Just got my diatom test slide!

#28 Post by Microscopy_is_fun » Sun Jan 02, 2022 11:49 am

josmann wrote:
Sun Dec 05, 2021 3:13 am
I went back at it today, this time with cross-polarized darkfield illumination. However, I kept my objective's iris fully open to maximize NA.

The lineolae on N. oblonga are now very highly contrasted. They are perceptually white/black when optimally illuminated. I have provided a detail view of a deformed stria. I feel like this is worth noting as it shows there is not a continuation of the grating pattern indicating some sort of false image.
Impressive images. Quite amazing how well you can resolve these tiny structures. Obviously you know how to get the most out of your equipment.

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