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Optimized Condenser-less dark field LED lighting

 
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Linden.g



Joined: 06 Apr 2009
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Location: PA USA

PostPosted: Mon Mar 30, 2020 6:18 pm    Post subject: Optimized Condenser-less dark field LED lighting Reply with quote

I thought I would try out a condenser-free darkfield set-up on an Olympus BH2 BHS as described by Kevin Webb https://www.researchgate.net/publication/265734017_Condenser-free_contrast_methods_for_transmitted-light_microscopy?fbclid=IwAR3JTEtkKd6rGHfvoBcAJxm82GcWPl8FRY1TNXG0j3rTzrYuh1p-q2nILyg. I placed two sets of LED ring lights, one between the stage and stage plate mount and one around the condenser swing top. I was trying to span the full width of the NA range. You can see the results below. The lower ring worked well for SPlanF2x, Splan 4x, Splan Apo 10x, Splan 20x. I tried to push the NA range up to 1.3 with the upper ring for D Plan Apo 20x UV, D Plan Apo 40x UV, Splan Apo 100x. I couldn't achieve dark field at the 100x. Lots of spiky flair as you go to the upper ring. All single shots. Unfortunately my diatom test slide came apart during the test.





















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Linden Gledhill http://www.flickr.com/photos/13084997@N03/


Last edited by Linden.g on Sat Apr 04, 2020 11:35 am; edited 1 time in total
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rjlittlefield
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PostPosted: Mon Mar 30, 2020 7:08 pm    Post subject: Reply with quote

Nice rig.

One issue I see is that each of those LED's is quite small, so while you're getting illumination from all points of the compass, the whole setup is still "partially coherent". This makes it vulnerable to an assortment of interference effects, more likely at higher NA. I would not be surprised if that's the cause of your spiky flair.

To reduce this effect, you might try adding some diffusion material, separated by a few mm from the LED dies.

The cleanest darkfield I've ever gotten was illuminating through a ring of ping-pong ball. That was at much lower mag than you're using. I've never tried it at high mag.

--Rik
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Lou Jost



Joined: 04 Sep 2015
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Location: Ecuador

PostPosted: Mon Mar 30, 2020 7:28 pm    Post subject: Reply with quote

Linden, that same paper inspired me to make a brightfield condenser-free setup using a half of a pingpong ball glued to the underside of a coverslip, with the whole assembly submerged, almost to the level of the coverslip, in a dilute solution of milk. I lit the milk with lasers. The milk decoheres the lasers and diffuses the light, and the pingpong ball creates a perfectly uniform light source similar in principle (though not as bright as) the LED hemisphere in that paper.
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Smokedaddy



Joined: 07 Oct 2006
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Location: Phoenix, Arizona

PostPosted: Mon Mar 30, 2020 8:19 pm    Post subject: Reply with quote

Looks like nice dark field to me Linden for non stacked images. As you know, all that can be easily cleaned up with PS. Just stay away from the 100x. Cool
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Linden.g



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PostPosted: Sat Apr 04, 2020 11:34 am    Post subject: Reply with quote

Thanks for the feedback. Rik, I took your advice and found a better LED light which gives a smooth diffuse light. It has helped to eliminate the flair spikes. The diatom images were taken with an SplanFl 2x and a Dplan Apo 40x UV. NA 0.85.






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rjlittlefield
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PostPosted: Sat Apr 04, 2020 11:41 am    Post subject: Reply with quote

Excellent!

Can you provide a link to buy that better LED ring?

--Rik
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Smokedaddy



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PostPosted: Sat Apr 04, 2020 12:45 pm    Post subject: Reply with quote

I absolutely knew you'd figure it out Linden. Cool
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Linden.g



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PostPosted: Sat Apr 04, 2020 1:48 pm    Post subject: Reply with quote

Thanks Jim and Rik, here you go https://www.ebay.com/itm/312733338679
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zzffnn



Joined: 22 May 2014
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Location: Boston, Massachusetts, USA

PostPosted: Sun Apr 12, 2020 11:27 pm    Post subject: Reply with quote

Great job, Linden!

How is your DF rig's diatom dotting ability at NA 0.85 40x?

If you can post a 100% crop on Pleurosigma angulatum, I can tell you how it compares with regular DF condener's dotting ability.

NA 0.85 is about as high as it gets for most oil DF condensers. At NA 0.85 DF, with a regular oil DF condensers, you should be able to resolve Pleurosigma angulatum to dots.

With a Leitz Heine variable DF / COL condenser (oiled) and a NA 0.95 LOMO 40x dry app objective, Frustulia rhomboides could also be resolved to dots. But Leitz Heine has a very slender and very large (variable) light cone.

I can gift you some diatom strew slides (message me if you want them). It is not cheap, nor convenient, to crash Klaus Kemp test slides Wink I have been there.
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Pau
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PostPosted: Mon Apr 13, 2020 12:41 am    Post subject: Reply with quote

zzffnn wrote:
...
NA 0.85 is about as high as it gets for most oil DF condensers. At NA 0.85 DF, with a regular oil DF condensers
...

With an oil 1.2/1.4 condenser you can get excellent DF with Na 1.0 oil objectives, at least in theory you could also do with NA 1.1 although I don't have such 1.1 objective.
But getting DF with a dry "condenser" and a 0.85 objective as Linden does is a big achievement, outside the range of typical DF dry condensers
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Linden.g



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PostPosted: Mon Apr 13, 2020 7:11 am    Post subject: Reply with quote

Thanks for the feedback. Here are some crops. The first is with Splan 40x NA 0.7 and the second with Splan Apo 40x NA 0.95




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Linden.g



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PostPosted: Mon Apr 13, 2020 8:09 am    Post subject: Reply with quote

I thought the Moiré pattern in these overlapping diatoms was interesting. 100% crop using Splan Apo 40x NA 0.95 with LED darkfield.


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Last edited by Linden.g on Mon Apr 13, 2020 8:15 am; edited 1 time in total
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zzffnn



Joined: 22 May 2014
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PostPosted: Mon Apr 13, 2020 8:10 am    Post subject: Reply with quote

Those look great there, Linden! Congratulations!

You have some big Amphipleura species on your strew slide and a NA 0.95 Apo. Why not give it a try by zooming in onto pixel level (to see how far your LED rig can go)?

Frustulia rhomboides would serve as better resolution target there (being raised and closer to the limit), though I am not sure you have it there.


Pau:

My previous comment (regarding not easy to go over objective NA of 0.85 even with oil DF condensers) came from my actual hands-on diatom dotting experience.

I used paraboloid and cardioid oil DF condensers (all marked 1.2-1.4) made by Zeiss Jena, Bausch and Lomb, and American Optical, with various (Apo or achromat) objectives made before 1980.

My experience is that DF objective NA above NA 0.85 does not always work. Yes, I did try a NA 1.1 objective, an iris objective of NA 0.7-1.0, a NA 0.95 objective, a few objective DF funnel stops of around NA 0.85, and a NA 0.75 objective.

AO produced darkfield funnel stops for their oil objectives / oil DF condensers and limited NA to right around 0.85-0.9. That is because DF image quality deteriorates quickly, by flare / mount (subject) thickness / optical property; you may get excellent DF image with Frustulia, but flare badly with Surirella.

Maybe modern Zeiss West high NA plan apos would work much better in DF.

I don't use dry DF condensers at all, as my DIY darkfield stops work reasonably well up to objective NA 0.65.
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Pau
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PostPosted: Mon Apr 13, 2020 9:23 am    Post subject: Reply with quote

0.95 is definitively outside the dry DF condensers range (the maximum NA of a dry system is 0.95 and NA of the objective must be smaller than the inner NA of the illuminating hollow cone, so it seems to me exceptional.

zzffnn,
I don't use modern objectives for microscope work. I was referring to a Leitz NPL Fluotar 50/1.0 oel, a very nice immersion objective, because I don't have diaphragm bundled high NA objectives I can't test further but my experience with classic equipment fully agree with classic papers.
My DF condensers are Zeiss 0.8/0.95 and 1.2/1.4 "ultracondenser"
I my limited experience both are better corrected that the peripheral zone of BF condensers with dark stops. The dry one is very nice for 40/0.70 or 0.75 objectives
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