Obtaining a clear image

Obtaining a clear image

Postby Stuart Bates » Thu Aug 30, 2012 8:43 pm

Hi all

As some of you may know, I acquired a microscope last Christmas (thank you Santa) and I am happy enough with looking at spores and taking approximate measurements etc...

The problem I am facing is trying to obtain a clear image of the "other bits" (basidia, cystidia etc...). I'm not sure if it's simply down to the fact that my microscope isn't exactly a high-end model or if it is just down to me being a numpty!

I have followed the "squash" method as best I can and proceed to view the sample, however, when I flick onto my x100 objective the image quality deteriorates very noticeably. I can't focus enough to get clear outlines on the various structures and I am finding the light source to be very unhelpful, I either have low light and a bit of fuzzy detail to look at OR a blinding light that drowns out any kind of detail :evil: I have attempted raising and lowering the condenser, opening and closing the Iris diaphragm and yet I can't seem to get a nice clear image at that level of magnification!! :cry:

Does anyone have any tips or advice that could help me with this problem?
Stuart Bates
 
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Re: Obtaining a clear image

Postby Leif Goodwin » Tue Sep 04, 2012 2:26 pm

There are explanations online for setting up the microscope, specifically the condenser, which is a very important component, far more so than most people realise. Search for 'kohler illumination'. You might also be using too much immersion oil, a mistake I made for a while.

That said, I'm not sure you need x1000 for basidia and cystidia, or at least I only use x400. The highest powers are good for examining and measuring spores. There are a few tricks, for want of a better word. Firstly get some needle nosed tweezers, and use them to pull off a tiny fragment of tissue from the area of interest. It is often important to know where the tissue comes from. Secondly, yes the squash method works well, but you often have to rack the focus in and out, and search like mad, to find what you are looking for. Sometimes it is easy peasy, but occasionally a fungus just does not want to play ball, or perhaps it is just not a good day.

If you do want to use x1000 on basidia, bear in mind that these structures are much larger than spores, and so the shallow depth of field becomes an issue. You can improve DOF by closing the iris diaphragm, at the cost of resolution, and image brightness.

I read that the late Peter Orton, a very talented mycologist, and an amateur, used a very modest microscope. I always recall that fact when I think I need a better microscope, and to be fair mine is decent enough, albeit some here have much nicer ones. :D
Fungi and Nature Photography: http://www.leifgoodwin.co.uk/Fungi/Fungi.html
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Re: Obtaining a clear image

Postby Stuart Bates » Tue Sep 04, 2012 4:50 pm

Hi Leif

Thanks for the reply... I have several questions in response.

1) I have tried to follow the instructions for Kohler Illumination in the past but they seems to imply that the microscope should have two seperate diaphragms (a field diaphragm and a condenser diaphragm). My microscope only has one on the condenser so I'm not sure that I will be able to utilise this method... Do you know if that is the case?

2) I usually use a single drop of immersion oil between the specimen and the objective... would you consider this to be "too much"?

3) I have some thin tweezers which I thought would be suitable, however, when you say "needle nosed" do you literally mean they are as thin as needles (or thereabouts)?

I think that's actually all of my questions for now... 8-)
Stuart Bates
 
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Re: Obtaining a clear image

Postby Mal Greaves » Tue Sep 04, 2012 8:37 pm

Stuart
You should be able to get as good an image through your 1000x lens as you do with the others and I think it is unlikely to be down to a non"high end" model. A well set up "Poor" scope will give better results than a badly set up "high end" one. If you are getting good images from your 400x then although the set up might not be spot on for the 1000x it should be close enough for acceptable images so I think it must be an immersion oil problem. When lenses are "parfocal" they should need no or very little adjustment when you change magnification if not (like mine) it can sometimes be difficult to focus on to a small item when you change the magnification.
Try this. Clean the 1000x lens of any surplus oil and using a slide with a good covering of spores work through the different magnifications including the 1000x without oil and see how much adjustment you have to make. Even with no oil you should be able to get a reasonable image. These are Scutellinia spores at 400 then 1000 no oil and finally 1000x oil
400b.jpg

1000b no oil.jpg

1000 oil.jpg


It was impossible to get any surface detail of the sopres without any oil
Try that and let us know what happens
Mal
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Re: Obtaining a clear image

Postby Stuart Bates » Tue Sep 04, 2012 9:25 pm

Hi Mal

Thanks for the advice, I will stick my camera against the eyepiece and take pictures of each magnification so you can see what I mean... My x1000 doesn't come close to the clarity of your images!! :cry:

All my other objectives provide good images so it is definitely something to do with the x100 objective I think.

Stuart
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Re: Obtaining a clear image

Postby Stuart Bates » Wed Sep 05, 2012 8:26 pm

As promised here are some pictures to "explain" what is happening with my scope.

I noticed that underneath my condenser there is a removable blue filter, is it best to keep this in place or not?

My images aren't great as I only have a basic point & shoot camera (Canon A3100) I have no control of the focus and therefore every shot is focusing on the micrometer rather than the specimen (spores from Geastrum triplex).

x40 magnification - at this level my images are perfectly clear (not that you can tell from the photo below)
Image

x100 magnification - again this is a clear level of magnification
Image

x400 magnification - starting to lose some clarity (not as much as shown in the pictures)
Image

x1000 magnification - this is about as focused as I could manage
Image

x1000 magnification with filter removed - slightly more detailed but still nowhere near what I would hope to see
Image

Anyone got any ideas? Would it be worth saving up for a "better" x100 objective or is it more likely to be something else causing my problem?
Stuart Bates
 
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Re: Obtaining a clear image

Postby Mal Greaves » Sun Sep 09, 2012 10:58 am

How do the photos compare with what you can see through the scope. It is often very difficult to get the same results on the camera compared to those you get when just looking through the lens.

Mal
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Re: Obtaining a clear image

Postby Stuart Bates » Sun Sep 09, 2012 3:17 pm

I would say that the pictures taken at x1000 are pretty true to the actual image. The pictures taken at lower levels of magnification are not as detailed as the true image produced.
Stuart Bates
 
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Re: Obtaining a clear image

Postby Stuart Bates » Sun Sep 09, 2012 9:36 pm

Just had another chance to get the microscope out... this time with a Leccinum scabrum that my friend "rescued" from our local golf course :lol:

I think the images are by far the clearest I have achieved with my microscope and the pictures below should show that... until you get to x1000 again!! There is some improvement but not much.

x400 magnification
Image

x1000 magnification
Image

Image

I suppose the images are definitely suitable for taking measurements so I should probably stop complaining at this point in time :D
Stuart Bates
 
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Re: Obtaining a clear image

Postby Mal Greaves » Mon Sep 10, 2012 9:28 am

Stuart
That's better, I would be happy with those shots.Did you do anything different on the setup?
Mal
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