Posted by
Csúcs Gábor-3 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-microscopy-problem-tp7584334p7584339.html
Dear Andre,
Currently I do not have any good ideas, but questions:
1) Is this 10-15 cycle dependent on the number of positions (locations) or the number of images (imaging speed)?
2) Do you see this modulation also if the stage movement is so small that you virtually stay at the same field of view?
3) Do you have a temperature control system?
Greetings Gabor
-----Original Message-----
From: Confocal Microscopy List [mailto:
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Sent: Montag, 12. Oktober 2015 10:35
To:
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Subject: Strange microscopy problem
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Dear confocalists,
we have a very strange microscopy problem, that is not directly related to confocal microscopy. Based upon the huge expertise on this list however, I hope that someone may have stumbled upon a similar problem or at least has some suggestions for the cause of the problem.
Currently we are trying to do widefield imaging of endogenous fluorophores (NADH, FAD). For this, we use a 365/50nm excitation filter and a 480/30nm emission bandpass.
When we try to do multiposition imaging (automated stage from Prior Scientific controlled by an old Optiscan controller) we observe a strange sinus-like modulation in the signal (ONLY with the mentioned UV excitation filterset) that has an amplitude of approximately 10-15% of the initial signal. Cycle time for the sinusoidal artifact is approximately 10-15min. This artifact ONLY appears, when the stage is actually in use (single-position imaging yields a stable signal). The whole setup is driven by the most recent Micromanager release which is (accept for the observed perturbation) working just fine.
So far, I can exclude an optical problem (imprecise repositioning of the stage and similar) - placing one of those fluorescent chroma-slides directly on the objective and repeating the experiments yields the very same results (sinusoidal artifact that vanishes when the stage is not in use). So far I have tried to attach grounding cables to virtually every piece of equipment including the mercury lamp and the camera (without any success in getting rid of this artifact).
Relevant equipment is a Nikon Ti-E with PFS (does not make a difference, whether it is in the lightpath or not), an Hamamatsu Orca ER-G CCD camera (exposure times are all in the range from 200-400ms) and a standard Nikon HBO with an Osram HBO103w/2 (tried changing it against the equivalent lamp from a Zeiss Axiovert200m without any changes).
Does anyone have any idea on how to tackle the problem? We are currently running out of ideas.
King regards
André
--
Dr. André Bernardini
Institute of Physiology
University of Duisburg-Essen
University Hospital of Essen
Hufelandstr. 55
D-45147 Essen, Germany
Ph ++49 201 723 4607
Fax ++49 201 723 4648
[hidden email]
www.uni-due.de/physiologie