Posted by
Andre Bernardini on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-microscopy-problem-tp7584334p7584343.html
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Hi Eric,
it is the second one (increase / decrease over the whole image in a
sinusoidal fashion).
Imaging interval is one minute. I did not try setting the stage to
really small xy-distances like Gabor suggested. However - it does not
make a difference, whether the stage is actually mounted to the
microscope or lying on the table beside it.
I already tried Steffen's suggestion with the other power circuit (with
all relevant devices) with no success. A z-Problem is also most unlikely
because I observe the problem also with the chroma-testslide lying
directly on the objective lens.
In the meanwhile it turned out, that it is indeed some kind of
electrical problem: I now provided proper grounding to the microscopes
filterwheel (the one with the cubes for epifluorescence) and the problem
seems virtually gone. I now observe a steady increase of fluorescence
signal which could very well have other (physical) explanations (some
kind of photoactivation / photoconversion in the chroma-testslide). I
will have a closer look at this tomorrow.
Does anyone now a reasonable explanation why a change in electric
potential of the filterwheel could cause such an error?
Anyway - thanks to all for your kind support!
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 16:21, Eric Marino wrote:
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> Andre,
>
> Is the sinusoidal artifact in a single image every 10-15 minutes (sinusoidal variation of intensity across the single image) or is it appearing in the time-lapse as an increase in intensity of the whole image every 10-15 minutes?
>
>
> Eric Marino
>
[hidden email]
>
>
>
>> On Oct 12, 2015, at 7:46 AM, Steffen Dietzel <
[hidden email]> wrote:
>>
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>> Andre,
>>
>> from what you are describing, probably either the excitation source or the detector or the z-position is fluctuating.
>> I would try the following:
>> - plug the mercury lamp into a different circuit, one that is not used by any other piece of this microscope, see if problem goes away.
>> - Try the same for the camera, if it has a separate power supply. And maybe for the xy.motor
>> - Do I read that correctly, that you don't see this fluctuation in green and red channels? not even a little in green?
>> - exchange excitation source to something other than HBO. With a chroma-slide, you can get plenty of fluorescence also with a halogen lamp attached to the fluorescence light path, with longer excitation .
>> - have you focused deep enough into the chroma-slide to exclude positioning problems in z? Do you keep the same focus over multiple exposures?
>> - it still could be a temperature problem, but with the z-position or with temperature of the camera or the lamp. Although the latter two seem unlikely, as long as you don't feel temperature changes on your skin. Maybe the motor itself causes heat that induces these changes. Do you have one of those big climate chambers? If so, try the Chroma slide thing again, but with a fully opened climate box, to allow heat dissipation.
>>
>> Good hunting
>>
>> Steffen
>>
>>
>> Am 12.10.2015 um 10:34 schrieb Andre Bernardini:
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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
>>>
>>>
>>> --
>>> ------------------------------------------------------------
>>> Steffen Dietzel, PD Dr. rer. nat
>>> Ludwig-Maximilians-Universität München
>>> Biomedical Center (BMC)
>>> Head of the Core Facility Bioimaging
>>>
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>>> D-82152 Planegg-Martinsried
>>> Germany