Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-microscopy-problem-tp7584334p7584340.html
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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
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany