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PostPosted: Sun Feb 10, 2019 8:31 pm 
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Posts: 233
Location: Idaho
This is one of the diatoms in Klaus Kemp's 8 form test plate, and the most difficult to resolve beyond the exterior shape. Over ten years ago I tried to see details inside with an oil immersion objective, but the best I could see was very faint hints of striae that might have been imaginary. I had already seen modern images on the internet.

A couple of days ago I tried again, armed with two sets of condenser stops I had bought on Ebay. I had also read on the internet that the striae, if not the punctae, should be visible to ordinary oil immersion objectives of 1.25 NA with an Abbe condenser and creative oblique illumination.

Trying different stops and both the Abbe and achromatic condenser, the best I could see was very faint hints of the striae. The achromatic condenser has smaller diameter lenses throughout, and the stops didn't work very well with it.

Then I decided to try the oil immersion dark field condenser:

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Dkfld_cndnsr.JPG
Dkfld_cndnsr.JPG [ 39.6 KiB | Viewed 191 times ]


The funnel stop is intended to be used with the oil immersion objective it's designed for, and I don't know if any of my objectives are the correct type. The main reason to not use the stop was an attempt to get circular oblique illumination around the very outer edge of the objective upper lens, to see if it would give greater contrast. After oiling the condenser to the underside of the slide and adjusting it just below touching the underside, and adjusting the centering screws, was able to get this narrow annular illumination ring while looking down through the eyepiece tube with the eyepiece removed.

This improved contrast a bit, but the striae were still extremely faint, and visible with only a couple of my oil immersion objectives. Then tried lowering the condenser focus and fiddling with the centering screws while looking through the 15X Compens eyepieces at the diatom. At some point the field and diatom got pretty dark, but contrast improved. Pulled one eyepiece and looked at the back lens of the objective. There was thin crescent centered at about 8 o'clock. Adjusted the centering screws and condenser focus until the crescent was very thin and centered around 6 o'clock, because the diatom was oriented vertically on the slide. The image was very dim, so turned up voltage selector switch on the transformer to maximum.

Wow! This worked! The striae now had enough contrast to be clearly visible with my best oil immersion objectives, and still faint but visible with the antique fluorite, which suffers from low magnification, and doesn't have quite enough resolving power to separate all the punctae in Frustulia Rhomboides as the other oil immersions can.

I spent a couple of days trying different combinations of old oil immersion objectives (all my objectives are old) and different illumination techniques. Early on I kept using the green filter, as it improved contrast and resolving power a bit in every test I tried.

Contrast of the striae got good enough I was able to get some pictures with the little eyepiece camera using the two best objectives. The camera was rotated so the orientation of the diatom was converted to horizontal so more could be imaged in the rectangular frame.

The first was made with my AO-Spencer 90X N.A. 1.30 apochromat:

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A.Pel_Apo_90x1.jpg
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The second image was made with my antique, uncoated Leitz Oel 1/12 100X N.A. 1.30. I think the image speaks for itself. I think it's not a coincidence this is my only other objective with a N.A. of 1.30. After sitting unused on an antique monocular stand for decades, it's going to get more use in the future.

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A.Pel_Leitz_1_12_1.jpg
A.Pel_Leitz_1_12_1.jpg [ 29.06 KiB | Viewed 191 times ]


Because of the hit-or-miss method I have to use to capture images from the little eyepiece camera, the image from the 90X Apo might not have been taken from the most exact focus. I was not able install the image capture software that came with the eyepiece camera, and the webcam image capture software that's in the Windows XP on the computer I'm using with the microscope isn't very advanced. The sample image visible for adjusting focus and image capture has much less resolution than the captured image, so I have to take a series of images while moving the fine focus a tiny bit at a time, hoping that one of the captured images will be at the best focus.

In GIMP I had to increase the image contrast from the 90X Apo a bit more than the antique Leitz. Don't know quite why it works this way, because the Apo gives a slightly sharper image with equal contrast when looking at stained bacteria slides.

And although the antique Spencer fluorite doesn't perform quite as well for this experiment, for some reason it has a bit less field curvature than any of my other old and antique oil immersions, so it's still handy for looking at stained bacteria slides and similar.

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Rick

A/O 10 Series Microstar
A/O 4 Series Microstar
A/O 4 Series Phasestar
A/O 4 Series Apostar
A/O Cycloptic Stereo
Several old monocular scopes in more or less decrepit but usable condition


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PostPosted: Mon Feb 11, 2019 8:30 am 
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Joined: Sun Dec 25, 2016 9:11 am
Posts: 745
Hi Rick,

thank you for your documentation of how to get this thing resolved!

I once got a bottle of water from the "Kraterquelle Bad Nenndorf", full of Amphipleura pellucida.
In the end my solution was similar to yours, a decentred cardioid condenser did it, even with a humble n.a. 1,25 achromat.
I proved to be important that the frustules are directly below the coverslip.
This might explain why your 90:1 apo was not as good as with the bacteria slides.

https://www.mikroskopie-forum.de/index.php?topic=28443.0

It is a bit difficult to accept that van Heurck made a much better photo 128 years ago. :oops:
http://www.baertierchen.de/feb2004.html


Bob


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PostPosted: Mon Feb 11, 2019 9:44 am 
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Location: Estepona
Nice work desertrat. Have not got very far with it myself.

MicroBob wrote:

It is a bit difficult to accept that van Heurck made a much better photo 128 years ago.
http://www.baertierchen.de/feb2004.html


Most impressive - here is an English translation ... and of course the formidable image he achieved!


Attachments:
Amphipleura pellucida.jpg
Amphipleura pellucida.jpg [ 165.67 KiB | Viewed 142 times ]

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PostPosted: Mon Feb 11, 2019 10:00 am 
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Joined: Sun Dec 25, 2016 9:11 am
Posts: 745
Van Heurcks flint glass slides and cover slips (Not to mention his arsenic based "yellow medium") would be difficult fo buy today.
I'm sure that many of them have been thrown away because their value wasn't obvious.
The same will have happend to most Nobert-test-plates. :evil:

But imagine 1891: A big business boom had just started - the low frame safety bicycle, even with air tube tires!
And then a microphotograph of this quality!


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PostPosted: Mon Feb 11, 2019 4:29 pm 
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Joined: Thu Apr 05, 2018 1:06 am
Posts: 233
Location: Idaho
Thanks, Bob and 75RR!

I forgot to mention in the first post that the oil immersion dark field condenser has one additional advantage. The illumination can be fairly easily adjusted between extreme oblique, and circular oblique illumination. The extreme oblique illumination from off one end of A. Pellucida does not give very good results trying to resolve the punctae of the other diatoms on the slide. When used to look at F. Rhomboides, only the striae can be seen.

When the condenser is adjusted for circular oblique illumination, the punctae on F. Rhomboides can be resolved.

_________________
Rick

A/O 10 Series Microstar
A/O 4 Series Microstar
A/O 4 Series Phasestar
A/O 4 Series Apostar
A/O Cycloptic Stereo
Several old monocular scopes in more or less decrepit but usable condition


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PostPosted: Mon Feb 11, 2019 5:15 pm 
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Joined: Mon Aug 21, 2017 9:02 pm
Posts: 1405
I believe, and others have shown, that a shorter wavelength will significantly improve the resolution. Theoretically, switching the light from green (550nm) to violet (400nm) would enhance resolution by ~38%. for this, one needs a good interference bandpass filter and intense illumination (a laser; a mercury lamp; there are potential dangers of course); Or, a more modern solution, a powerful 400nm LED.

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Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo


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PostPosted: Mon Feb 11, 2019 6:32 pm 
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Joined: Sun Dec 25, 2016 9:11 am
Posts: 745
Hi Doron,
Peter Höbel used near UV light to resolve Amphipleura p.:
http://www.mikroskopie-ph.de/index-Amphipleura.html

The upper left image shows about what is possible this way.

Bob


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