First diatom test...what went wrong?

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Free2Fish
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Location: Manitoba, Canada
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First diatom test...what went wrong?

Post by Free2Fish »

I tried my first diatom test using Klaus Kemp's 8 form test slide and got some pretty bad results. I've posted images and my analysis to a blog I've just started and if anyone has the time I'd welcome any ideas on how to fix this.

http://micro-nut.blogspot.ca/

Cheers,
Harry
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

Pau
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Location: Valencia, Spain

Post by Pau »

Harry,
the Zeiss Neofluar are excellent objectives. With my 40 0.75 I can easily resolve Pleurosigma punctuations, both in BF, DF, DIC and PhC.

Your first picture is clearly misfocused or vibration blurred. Are you sure that you use silent mode with your 40D?. Do you have any source of external vibration? (when you tether the camera to show live image at the computer screen as you do it's very easy to see, and you can get accurate focusing if you have any parfocality error in your camera setup).
To get good image of not moving subjets is much better to set ISO 100 and longer exposure to avoid noise.
Can you visually resolve the diatom puctuations?. If yes as it seems the problem must be at the camera/technique side and not at the objectives.
Also check the condenser heigh and aperture, if too low or too closed you lost a lot of resolution.

Please post detailed pictures and description of your camera adapter if you want to see discussion about it. Is it parfocal with the eyepieces?. You say that you have problems to focus the specimen, an this sounds very strange for me if the system is well set up.
Hope this may help.
Pau

Free2Fish
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Joined: Fri Oct 05, 2012 8:47 am
Location: Manitoba, Canada
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Post by Free2Fish »

Thanks for your comments, Pau.

Yes, the camera is set to silent mode. I just checked and am now trying Mode 2 to see if that makes a difference.

There is no clear source of external vibration but that is not to say the setup is solid. The table I'm using isn't great but seems stable. If all else fails I can move the scope to a solid table that I have a basement mill set up on but this is only a last resort since I want to be doing this upstairs.

The ISO was set at 100.

A thorough description of the camera setup is detailed in the previous post on the blog. As stated, it's not the best setup for my 40D but will have to do until I can find a better solution. I have followed your posts on what you are using and am looking for something similar.

Visually, I can see the punctae readily with the 40X when looking through the microscope oculars and I can just make them out with the 25X. I have trouble seeing them on the laptop screen in EOS utility (the latest version) with either objective. This is where I have trouble focusing the image, the punctae just don't become clear whereas focusing through the eyepiece is accurate and easy. I can say the computer view is parfocal with the eyepiece view but I'm just not sure since I can't focus the computer image as sharply as the eyepiece image.

The condenser was set up for Koehler illumination but it only has a 0.63 NA. I just tried the same test just now with a 0.90 NA condenser and results seemed better but the photos didn't resolve the punctae either.

Regards,
Harry
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

Pau
Site Admin
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Joined: Wed Jan 20, 2010 8:57 am
Location: Valencia, Spain

Post by Pau »

What kind of optics are inside your adapter?
If you have an adequate tripod or copy stand try to take pictures afocally with a camera lens (like a standard 50mm or even a kit zoom at 40mm wide open) trough a viewing eyepiece. Even with a powerfull lightsource you can try to take pictures hand held.

I don't think the condenser lens would be the cause: 0.63 may limit very little the aperture for a 0.75 objective, but if you have a 0.9NA it may be more adequate. In fact in PhC the actual aperture of the phase ring will be smaller and for BF you usualy need to close the aperture to get enough contrast.
Pau

Free2Fish
Posts: 31
Joined: Fri Oct 05, 2012 8:47 am
Location: Manitoba, Canada
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Post by Free2Fish »

I have no idea what kind of optics are in the adapter. What I did do was take your advice and shoot a picture through the eyepiece. I set up an old point and shoot on a tripod and took this picture. I guess one difference is that a different 0.90 NA condenser was used but as you said that shouldn't make much difference. I guess from this I can take that my camera setup is to blame.

Image
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

Free2Fish
Posts: 31
Joined: Fri Oct 05, 2012 8:47 am
Location: Manitoba, Canada
Contact:

Post by Free2Fish »

Pau wrote:Please post detailed pictures and description of your camera adapter if you want to see discussion about it.
I've taken pictures of the camera adapter in case they help. The two separate pieces are the adapter and a Z Clamp to fit it to the Universal. I've found almost nothing on the internet about this unit so if someone can shed light on this issue, please jump in.


Image

Image

Image
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

Pau
Site Admin
Posts: 6053
Joined: Wed Jan 20, 2010 8:57 am
Location: Valencia, Spain

Post by Pau »

Harry,
I see a good improvement (mainly at your blog...not very practical to follow the thread in two sites).
The 0.60X adapter must be intended for C mount small sensor cameras. 1.2X is much more reasonable. I thought your images where taken at high ISO because they where heavily cropped and sharpened (ie more noise).
Another possible issue (not with green light) is the need of eyepiece corrections because Zeiss objectives aren't fully corrected. I don't know if the optics in your phototube may or may not perform the required corrections. You can test it with a high contrast slide looking for radially symetrical color bands at high contrast limits absent at the image center and increasing towards the periphery.

The ideal phototube for your scope will be this model:
http://www.ebay.com/itm/Zeiss-Microscop ... 27cf7a0d30
paired with a kpl eyepiece and a relay lens over it. My 63mm 0.25X adapter is hard to find (and often expensive) but you may adapt a MC63 camera tube with some brico.
Pau

Free2Fish
Posts: 31
Joined: Fri Oct 05, 2012 8:47 am
Location: Manitoba, Canada
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Post by Free2Fish »

Hi Pau,

Sorry for the inconvenience. I had already posted twice in response to your suggestions and was reluctant to post a third before receiving an answer.

I've sent an email to Diagnostic Instruments to ask about any correction in this lens and will wait to see if they answer.

I'm trying to picture how to use the photo tube you suggest I get. I guess the Kpl eyepiece goes into the top of the tube and the tube fits into the Universal camera port. The where does the relay lens go and how is the camera attached?

Harry
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

Pau
Site Admin
Posts: 6053
Joined: Wed Jan 20, 2010 8:57 am
Location: Valencia, Spain

Post by Pau »

Free2Fish wrote:I'm trying to picture how to use the photo tube you suggest I get. I guess the Kpl eyepiece goes into the top of the tube and the tube fits into the Universal camera port. The where does the relay lens go and how is the camera attached?
Yes, the system will end just as mine, the phototube I linked is a focusable (and lockable) empty tube with a Z dovetail at the base and an eyepiece fit at the end. the main body diameter is 40mm and over it fits both my camera adapter or the MC63 (that is similar but with a magnetic shutter and an specific camera mount for a dedicated Zeiss camera).
Pau

Free2Fish
Posts: 31
Joined: Fri Oct 05, 2012 8:47 am
Location: Manitoba, Canada
Contact:

Post by Free2Fish »

Thanks Pau, that's the exact information I was looking for.

I found out yesterday from Diagnostic Instruments the adapter I have doesn't have any correction in it so I assume that has affected my images to some degree. How much I guess I can't know at this point.

Now it's a matter of poring over eBay posts till I find something that won't break the bank. I now understand why people purchase
a turnkey setup. :)

Harry
Zeiss Universal
Leitz Laborlux S
Canon 40D
Canon T3i
http://micro-nut.blogspot.ca/

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