Posted by
Joao Lagarto on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-microscopy-problem-tp7584334p7584342.html
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Hi André,
The heated incubator does not rule out a temperature effect on its own. It will mostly depend on the temperature controller. If e.g. this operates on a periodic on-off switch, you can get fluctuations in the temperature that may affect the fluorescence signal. To rule out a temperature artefact, and given that both NADH and FAD fluorescence vary linearly with temperature, you could measure the temperature of the stage during the acquisition and try to correlate it with the fluorescence signal.
Good luck,
João
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Andre Bernardini
Sent: Monday, October 12, 2015 10:28
To:
[hidden email]
Subject: Re: Strange microscopy problem
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Hi João,
thank you for your suggestion.
I can exclude a temperature-artifact. The sample resides in a heated on-stage-incubator (37°C).
Also a chroma-testslide, that lies directly on the objective, gives the very same artifact (doesn't matter whether on-stage-incubator heating is on or off), making a temperature-effect most very unlikely.
Kind 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
On 12.10.2015 11:17, Joao Lagarto wrote:
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>
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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:
[hidden email]] On Behalf Of Andre
> Bernardini
> Sent: Monday, October 12, 2015 9:35
> To:
[hidden email]
> Subject: Strange microscopy problem
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
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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
>
>
>
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