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 Post subject: Radiolaria observations
PostPosted: Thu Jan 10, 2019 10:35 am 
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These mounting experiments were inspired by the recent post
viewtopic.php?f=10&t=6725&p=61286&hilit=radiolaria#p61286

These are cleaned Radiolaria from Barbados, kindly donated by forum member MicroBob. Thanks Bob!

I mounted them in NOA61 glue, by a modified (simplified, for me) protocol:

The stored glue from the fridge was let to reach room temperature. I then laid a drop, diamater about 4mm, on the 22x22mm coverslip. The coverslip was heated at 50-70C for about a minute on a pre-heated stainless steel flat block. The glue drop spread due to the lowered viscosity. Radiolaria powder from the sample bag was sprinkled onto the coverslip by means of a thin painter's brush. The coverslip was covered with a slide. The slide was picked off the heating block and was cured under an "atmosphere" UV lamp for an hour.

Photos were taken with a 25X/0.45 phase contrast and a 40X/0.75 Neofluoar phase contrast objectives, respectively. Air bubbles were rare.

Below are brightfield single images, resized and cropped but otherwise untouched, to demonstrate the visibility. Sizes in the pic titles are approximate width of specimen. I expect to post more photos later.


Attachments:
(1) BF 40X   52micron.jpg
(1) BF 40X 52micron.jpg [ 98.5 KiB | Viewed 1324 times ]
(2) BF 40X   50micron.jpg
(2) BF 40X 50micron.jpg [ 99.28 KiB | Viewed 1324 times ]
(3) BF 40X   155micron.jpg
(3) BF 40X 155micron.jpg [ 157.91 KiB | Viewed 1324 times ]
(4) BF 25X   200micron.jpg
(4) BF 25X 200micron.jpg [ 98.85 KiB | Viewed 1324 times ]

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PostPosted: Thu Jan 10, 2019 12:20 pm 
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Added are some darkfield photos. Single images, resized only. 40X/0.75 Neofluar, turret phase condenser (position "D") oiled to slide.


Attachments:
(5) DF.JPG
(5) DF.JPG [ 134.51 KiB | Viewed 1313 times ]
(6) DF.JPG
(6) DF.JPG [ 103.81 KiB | Viewed 1313 times ]
(7) DF+blue filter .JPG
(7) DF+blue filter .JPG [ 86.57 KiB | Viewed 1313 times ]
(8) DF.JPG
(8) DF.JPG [ 114.04 KiB | Viewed 1313 times ]
(9) DF+blue filter .JPG
(9) DF+blue filter .JPG [ 116.22 KiB | Viewed 1313 times ]

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PostPosted: Thu Jan 10, 2019 12:43 pm 
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Finally, phase contrast with a blue filter photos, stacks of 10-20 images, spaced at 1 micron distances, 40X/0.75 Neofluar Ph2 objective.
Stacks were tweaked in microsoft picture manager to enhance contrast.


Attachments:
(10) Phase contrast stack.jpg
(10) Phase contrast stack.jpg [ 38.45 KiB | Viewed 1310 times ]
(11) Phase contrast stack.jpg
(11) Phase contrast stack.jpg [ 76.48 KiB | Viewed 1310 times ]
(12) Phase contrast stack.jpg
(12) Phase contrast stack.jpg [ 43.84 KiB | Viewed 1310 times ]
(13) Phase contrast stack.jpg
(13) Phase contrast stack.jpg [ 96.61 KiB | Viewed 1310 times ]

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PostPosted: Thu Jan 10, 2019 2:52 pm 
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Fine images!
Makes me want to get hold of a Radiolaria slide and try my luck. :)

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PostPosted: Thu Jan 10, 2019 3:47 pm 
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Hi Doron,

nice images! Since radiolaria have no colour you might just use one colour channel (I think green is best) of your image so you effectively come close to the image quality of an apochromat. I like to use round cover slips for permanent slides because it is much easier to get a good looking slide with them. Octagonal cover slips would be nearly as good but probably cheaper to make but I have never seen them.
I once bought chinese round cover slips from ebay (what can be wrong with them ? :D ) but they were complete rubbish as the were up to 0,25mm thick. :evil: I got mine from a woman at the german forum, originally bought from some lab supplier.

Bob


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PostPosted: Thu Jan 10, 2019 5:48 pm 
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Location: Idaho
I like those images, especially the last two from the first image set. I have an old Perfect brand prepared slide of pond plankton made in the '60s, and one of the objects looks a lot like those last two images. Are radiolaria found in fresh water?

MicroBob wrote:
Hi Doron,

nice images! Since radiolaria have no colour you might just use one colour channel (I think green is best) of your image so you effectively come close to the image quality of an apochromat. I like to use round cover slips for permanent slides because it is much easier to get a good looking slide with them. Octagonal cover slips would be nearly as good but probably cheaper to make but I have never seen them.
I once bought chinese round cover slips from ebay (what can be wrong with them ? :D ) but they were complete rubbish as the were up to 0,25mm thick. :evil: I got mine from a woman at the german forum, originally bought from some lab supplier.

Bob

That got me to thinking, that I've never checked the thickness of the 1400 Chinese made 18mm round coverslips that I bought about 10 years ago from an Ebay seller of Chinese microscopes and accessories based in southern California. At that time, 1000 of these coverslips cost about $30. Now the price is over double that from any source I can find.

I got out my trusty old Brown & Sharpe vernier micrometer, that measures to .001 inch with the vernier scale giving readings of .0001 inch. I pulled out about 8 of these coverslips from the little plastic box and measured them. They varied from .0058 to .0062 inches, or roughly .15 to .16 mm.

Checking again, it looks like two purchases were made from different sellers, most of the cover slips are "Sail" brand, and a few boxes are "Pearl" brand. I think the ones I checked were Sail brand. Boxes from both brands show a thickness range from .13 to .17mm.

Most of the slides I've mounted with these coverslips are viewed with the lower power objectives, seldom higher than 20x. I guess it would be a good idea to check the thickness of any coverslips that will be used for high power observation.

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


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PostPosted: Thu Jan 10, 2019 8:16 pm 
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Thanks 75RR, MicroBob and desertrat!

MicroBob wrote:
...Since radiolaria have no colour you might just use one colour channel (I think green is best) of your image so you effectively come close to the image quality of an apochromat. I like to use round cover slips for permanent slides because it is much easier to get a good looking slide with them.
Yes. Green light is considered best for phase contrast because they designed the optics for a wavelength of green (about 550nm). If I find a sufficiently narrow band green filter I will give it a try.

In fact, I have a 40X/1.0 Planapo objective with iris, but it is oil immersion; I might close the iris down to an NA of 0.7-0.8, and compare it, without immersion, with the 40X/0.75 Neofluar! the latter is an old and I suspect somewhat delaminated of late...

Round coverslips I save for the next run, hoping it will be free of debris and bubbles.

desertrat wrote:
I guess it would be a good idea to check the thickness of any coverslips that will be used for high power observation.
. Yes, my Mitutoyo 0.001" mechanical caliper approves of my coverslips, but I will double-check with a more accurate micrometer.

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PostPosted: Thu Jan 10, 2019 8:24 pm 
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Hi Doron,
in bright field you can just use white light and take the photo. Then just use one colour channel in the image editor and convert to gray scale. I get somewhat better diatom images this way.

Bob


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PostPosted: Thu Jan 10, 2019 8:26 pm 
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Hi Bob,
A good idea, thanks!

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PostPosted: Thu Jan 10, 2019 8:32 pm 
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I used the vernier mic mainly to see how much variation there was. I think a .001" caliper would be fine for all but the most exacting requirements. If the covers are between .006" and .007", they should be good for most mounts. If any extra thick covers show up during checking, I think they would be OK for thick mounts that will only be viewed with low power objectives.

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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: Fri Jan 11, 2019 8:53 pm 
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In the previous part I presented photos taken with a 40X/0.75 phase contrast Nefluoar objective. A significant chromatic abberation is shown. At least some of it might perhaps be attributed to refractions within the Radiolaria "glass" structures (If anyone knows otherwise, please comment). Following MicroBob's comment, I thought it might be of interest to compare the Neofluar with a Planapo objective. The former is expected to be better, but by how much would it matter for the Radiolaria ? I have a 40X non-phase iris oil objective, so gave it a try.

Here is a set of darkfield and brightfield images. All taken with 40X/1.0-0.6, 160/- Planapo oil immersion objective. I closed the iris to reduce the NA from 1.0 to about 0.7-0.8 (there are no graduation marks on the objective, so this is approximate). I oiled the objective to the slide as well as the condenser to the slide. The exact thickness of the coverslip does not matter in this case.

All photos except No 14 are single images.
Comparing brightfield photo No 18 to photos No 1-4 above (in the initial post), and darkfield photos No 14-17 to photos No 5,6,8 above shows the superiority of the Planapo for both BF and DF in this case.

I did not take any photo with the Planapo objective dry, without immersion oil, since visually it seemed to be inferior.


Attachments:
(14) Planapo 40X -ca.0.7 Oil (stack of 13).jpg
(14) Planapo 40X -ca.0.7 Oil (stack of 13).jpg [ 50.62 KiB | Viewed 1208 times ]
(15) Planapo 40X -ca.0.7 Oil .JPG
(15) Planapo 40X -ca.0.7 Oil .JPG [ 154.92 KiB | Viewed 1208 times ]
(16) Planapo 40X -ca.0.7 Oil .JPG
(16) Planapo 40X -ca.0.7 Oil .JPG [ 132.43 KiB | Viewed 1208 times ]
(17) Planapo 40X -ca.0.7 Oil .JPG
(17) Planapo 40X -ca.0.7 Oil .JPG [ 106.18 KiB | Viewed 1208 times ]
(18) Planapo 40X -ca.0.7 Oil .JPG
(18) Planapo 40X -ca.0.7 Oil .JPG [ 127.42 KiB | Viewed 1208 times ]

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PostPosted: Fri Jan 11, 2019 9:35 pm 
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Hi Doron,
I took you image from the neofluar and used the green colour channel to get a b/w image and sharpened a bit. For me this looks better than the colour image.

Bob


Attachments:
Doron_rad1.jpg
Doron_rad1.jpg [ 62.86 KiB | Viewed 1203 times ]
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PostPosted: Fri Jan 11, 2019 10:22 pm 
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Hi Bob,
I immitiated your processing: fed the image into ImageJ, split channels, and both the green and red channels are indeed better (more contrast, appear sharper) than the blue channel.

Likewise, had I used monochromatic light, I would have gotten a similar result even without post-processing.

But, there can be an alternative argument: Because of the appearance of the original photo, say the Neofluar, we know that the Radiolarian is indeed transparent! like a piece of broken glass, it disperses light. Of course, based on previous experience, we were sure to start with that it is trasparent; yet...
Hope to take some photos withh the Planachromat tomorrow and see how it looks like.

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PostPosted: Sat Jan 12, 2019 10:31 pm 
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As an appendix to the above, I took three photos of a Radiolarian fragment, mounted in NOA61, with my three Zeiss 40X objectives:
40X/0.75 Neofluar Ph2
40X/0.65 Planachromat
40X/~0.8 Planapo Oil immersion (it is the iris objective, NA 0.6-1.0).
Brightfield, the illumination and the custom white balance in the camera were kept constant. All three objectives are parfocal to within a few microns.
Focusing was manual, on the same region of the specimen, and was based on the x5 magnification feature of the camera LCD screen.
All photos are single images, and all were brightened, resized and cropped by the same amount in post-processing.
The results shows that the Planachromate was of the least sharpness, Neofluar phase contrast was better, and the Planapo came first. While these results could be expected, they visually demonstrate the effects of chromatic aberrations on images of glass-like transparent specimens, which might themselves (perhaps) disperse colors by refraction.


Attachments:
40X-0.65 Planachromat.jpg
40X-0.65 Planachromat.jpg [ 66.29 KiB | Viewed 1148 times ]
40X-0.75 Neofluar Ph2.jpg
40X-0.75 Neofluar Ph2.jpg [ 68.82 KiB | Viewed 1148 times ]
40X - ~0.8 Planapo oil immersion.jpg
40X - ~0.8 Planapo oil immersion.jpg [ 69.96 KiB | Viewed 1148 times ]

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PostPosted: Sun Jan 13, 2019 8:55 am 
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Hi Doron,
nice comparison with interesting results!
The image taken with the Planachromat looks quite bad, and different from the two others. It's possible that the image doesn't quite represent the usual performance of the objective in good condition.
The Planapo really shines - it was a tremendously expensive objective when new and still offers great performance. Very comfortable too: No image editing necessary for a perfect image! I think a normal brightfield Neofluar would have been even better than the PH one.

I once read that a manufacturer described the performance of his fluorite and apo objectives like this: Fluorite has no colour error in the image plane, apo has not colour error in image plane and in the depth of the image. This would expain the coulour in the very 3D radiolaria image taken with your fluorite.

In the center of the image and with a flat, thin object all three correction classes should perform the same with the same n.a..

The glass like material is difficult to detect in transmitted light images. A while ago I showed an image on dark ground with illumination from the side. Here the radiolaria were clearly visible as glass like.

Bob


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PostPosted: Sun Jan 13, 2019 1:33 pm 
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MicroBob wrote:
... I think a normal brightfield Neofluar would have been even better than the PH one.
Well. I thought the same, so I took a picture of the same specimen under the 40X/0.75 Neofluar (non phase-contrast) vs the 40X/0.65 Plan. The results were the same as before: The non-PC Neofluar yielded a sharp image, with CA, similar to the PC Neofluar; the Plan gave the slightly blurred image as before. It is possible that my results are not very representative, but such objectives are quite widespread (to judge from the high number of users of Zeiss Standards alone) and they are all aged somehow.
Quote:
...A while ago I showed an image on dark ground with illumination from the side. Here the radiolaria were clearly visible as glass like.
I searched for posts on this forum where the image would be expected, but found none. Please provide a link to it. Thanks.
Doron

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PostPosted: Sun Jan 13, 2019 5:27 pm 
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Here two quick stacks, taken with a normal objective 6,3:1 or 10:1, illumination from the side with a Ikea Jansjö lamp and a piece of wheelbarrow inner tubing as background. I was very surprised how beautiful this special material was. They are radiolaria from deep sea, obvioulsly cleaned very carefully, regrettably not my material. I borrowed it for a group meeting and the members very very happy with the resulting slides.

Bob


Attachments:
Radiolarien 1 fertig 1024.jpg
Radiolarien 1 fertig 1024.jpg [ 163.51 KiB | Viewed 1080 times ]
Auschnitt 1 1024.jpg
Auschnitt 1 1024.jpg [ 185.36 KiB | Viewed 1080 times ]
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PostPosted: Sun Jan 13, 2019 6:17 pm 
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Well done, Bob! superb looking micro-structures.

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PostPosted: Sun Jan 13, 2019 10:09 pm 
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Excellent images and beautiful subjects!

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PostPosted: Mon Jan 14, 2019 4:33 am 
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Hi Guys I am under the impression that what I have seen in the photos are not all radiolarians but some spicles.
I will post a couple of my photos of radiolaria.
Radiolaria come under the name of Polycystina.


Attachments:
PICT2000-004.jpg
PICT2000-004.jpg [ 49.47 KiB | Viewed 1048 times ]
PICT1999-002.jpg
PICT1999-002.jpg [ 75.99 KiB | Viewed 1048 times ]
12227063_546605515514387_1866105870427273304_n.jpg
12227063_546605515514387_1866105870427273304_n.jpg [ 48.05 KiB | Viewed 1048 times ]

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Thank you :shock:
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exmarine :x

uses Watson 'Service' 1950 compound.
uses Watson Stereo 1960 ish.
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