# Axial resolution of microscopic techniques

Dear,
I am a PhD student at the Catholic University of Leuven (Belgium) in material sciences with a question about microscopy.
One of my collegues is exploring the capabilities of a CSLM (confocal scanning laser microscope) to observe in-situ the high temperature behaviour of refractory materials. The material is not transparent to the laser light, so he can only see the surface of the sample. He is using the cslm because our institute does not have a optical microscope with a heating stage.
A problem he faces is that the image quality deteriorates quite rapidly when the sample reaches high temperatures (1600°C). The explanation we are thinking of, is that the sample roughens during the experiment. Measurement has shown that before the tests the roughness was about 0.2µm; after the tests is was 5µm. The small focus plane of the CSLM could thus cause blurring of the image.
In order to elaborate this explanation, we would like to have an idea about the axial (depth, vertical) resolution of the CSLM equipment and other imaging techniques (LOM, SEM). Can someone please suggest a good reference where we can find such data?
Regards,
Dirk
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Dear,
I am a PhD student at the Catholic University of Leuven (Belgium) in material sciences with a question about microscopy.
One of my collegues is exploring the capabilities of a CSLM (confocal scanning laser microscope) to observe in-situ the high temperature behaviour of refractory materials. The material is not transparent to the laser light, so he can only see the surface of the sample. He is using the cslm because our institute does not have a optical microscope with a heating stage.
A problem he faces is that the image quality deteriorates quite rapidly when the sample reaches high temperatures (1600°C). The explanation we are thinking of, is that the sample roughens during the experiment. Measurement has shown that before the tests the roughness was about 0.2µm; after the tests is was 5µm. The small focus plane of the CSLM could thus cause blurring of the image.
In order to elaborate this explanation, we would like to have an idea about the axial (depth, vertical) resolution of the CSLM equipment and other imaging techniques (LOM, SEM). Can someone please suggest a good reference where we can find such data?
Regards,
Dirk

Dirk, Take a look at a good educational web site like Carl Zeiss. Microscope maker sites usually have tons of user info. The depth of focus relates to the NA and distance from the focal plane. This is not altered by being a confocal. The greater the NA the less the dept of focus. I would suspect that you are using very LWD objectives, you might want to consider that the air between the specimen plane and objective is becoming quite hot and this is distorting the image.
Thanks,
Kevin Cunningham SMS
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If it is indeed a large increase in surface roughness which is causing the drop in image quality, it might be worth trying one of the image stacking programs such as Helicon Focus or Combine Z(?). You may then be able to combine a stack of images with different focus. Note however that they can produce artefacts if they get confused, so you may need to "correct" by reference to a manual examination of the focus planes.
David
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David Littlewood

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Kevin Cunningham wrote:

Not to mention melting the cement in the objective lens! Has the OP's colleague told the owner of the microscope what he's doing to it???
Nobody would be allowed to do that to my microscope, thanks very much.
I suspect thermal expansion and/or thermal lensing in the objective lens.
Cheers,
Phil Hobbs
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Thank you all for the fast reply.
Maybe it is best to give some more information about my problem. On http://perswww.kuleuven.be/~u0049826/ you can find three cslm images of the refractory material. The idea is to study the dissolution of one phase in the other as a function of time. ( So building a 3D-image from different layers is not really an option...)
However, we find that 1) the image quality between room temperature measurements and high temperature differs strongly (blurry and much darker) and 2) the image quality even further deteriorates when we stay at high temperatures. We would like to find an explanation for both as this limited image quality makes the analysis much more difficult.
The blurring effect during heating can possibly be explained by the lensing effect of the gas between the sample and the objective lens. However, if the temperature profile of the gas around the sample is stable one should be able to correct this effect, no? If it is not stable, does this mean that all high temperature LOM techniques have this drawback?
The darkening of the image could possibly be due to roughening of the sample due to differences in thermal expansion between the phases. If in cslm an object is not exactly in the focus plane its intensity drops rapidly. Does this seem a reasonable explanation?
The fact that the image becomes even more blurry when we stay at high temperature could be explained by further roughening due to the reactions taking place.
Please comment, all input is welcome...
Greetz,
Dirk
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Well, I'm a chemist (ex) not a metallurgist, and the pictures are *very* small to get a good impression of what is going on, but FWIW:
(1) The darkening on heating could be due to some kind of oxidation. I know you said it was a refractory, not a metal, but you did not say what it was.
(2) Alternatively, could it be some volatile in the heating chamber which is decomposing on the surface?
(3) The slight degradation of the sharpness looks more like a change in the sample rather than a change in the optics - though the image is way too small to make judgements.
(4) Could the specimen be partially softening or melting? Or absorbing O2 or something from the atmosphere.
(5) Finally (as I think someone else hinted at) are you sure the heat is not affecting the optics of your microscope? Does it still give sharp images after cooling?
((6) Could you put some reference material in the chamber, which you know is entirely unaffected by the temperature - if this still images sharply, but the specimen does not, then you should be fairly certain it is a specimen/temperature effect, not a microscope/temperature effect.
Hope these may at least trigger some useful lines of enquiry.
David
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David Littlewood

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David Littlewood wrote:

Also - thermal expansion of the sample may cause problems if you're not refocusing. We used a special "microscopic" telescope at a distance of ~0.5 meters to observe glass melting many years ago. Gregg
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Some very interesting remarks were made. I'm trying to get the original images (910x525 pixels) but for now these smaller ones will have to do.
David, the sample is a magnesia-chromite refractory. The bright phase is MgO.Cr2O3 spinel; the darker phase MgO. The sample was heated in an Argon flow. Oxidation and gas formation seem not possible to us, seen the nature of the material. Possibly some silicate phases are present which might melt at that temperatures. However, their amount is probably too small to have an effect. We believe that the heat is not effecting the optical set-up of our cslm on a long term. When a new sample is inserted, good quality images can be obtained without adjusting lenses etc. We did not examine the samples after heating with the cslm, but with SEM-BSE. The quality of these BSE images were not good (certainly compared with the images taken before heating in cslm), which supports our believe that the sample roughened during the experiment.
To react to Gregg, we did refocus to account for the thermal expansion but we were not able to get an equally good image as at room temperature. That's is exactly what we are trying to explain.
Best,
Dirk
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Dirk wrote:

Can you put a small polished (99.9%) Al2O3 part in your field of view - or Pt? At 1600 C you'll most likely get some grain growth in the Al2O3 but you might get a clear image which would rule out optical effects (or not) My only thought is to image the sample at different (depths) focal planes and combine the focused images as mentioned earlier. ImageJ has some plugin's that can do that and it's free.
Good luck Gregg
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wrote:

What did you find out from the MSA listserver? Was that insufficient?
Gary Gaugler, Ph.D. Microtechnics, Inc. Granite Bay, CA 95746 916.791.8191 gary@microtechnics dot com
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