http://confocal-microscopy-list.275.s1.nabble.com/Strange-microscopy-problem-tp7584334p7584336.html
I can exclude a temperature-artifact. The sample resides in a heated
Dr. André Bernardini
Hufelandstr. 55
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> Hi Andre,
>
> Can it be a temperature artefact? Both NADH and FAD autofluorescence are temperature dependent.
>
> Regards,
> João
>
> --
> João Lagarto, Ph.D
> Instituto Gulbenkian de Ciência,
> Oeiras, Portugal
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
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> Sent: Monday, October 12, 2015 9:35
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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
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