Strange microscopy problem

[Lathrop, Kira L]

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Dear Andre,
We have the same microscope and the problem may be tied to the stage motion, shutter speed and computer speed.  We ran into a similar thing, and fixed it by programming a slight delay to allow the shutter to (reliably) open fully before beginning acquisition in the new position or by adding another channel (black or brightfield) before your fluorescent channel.
Kira
___________________
Kira L. Lathrop, MAMS
Assistant Professor
Co-Director, OVSRC Imaging Module
University of Pittsburgh
School of Medicine, Department of Ophthalmology
Swanson School of Engineering, Department of Bioengineering
203 Lothrop Street Room 1030
Pittsburgh, PA 15261
[hidden email]
412.647.3492

>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Andre=20
>> Bernardini
>> Sent: Monday, October 12, 2015 9:35
>> To: [hidden email]
>> Subject: Strange microscopy problem
>>=20
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>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your =

posting.
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>>=20
>> Dear confocalists,
>>=20
>> 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.
>>=20
>> 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.
>>=20
>> 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.
>>=20
>> 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).
>>=20
>> 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).
>>=20
>> Does anyone have any idea on how to tackle the problem? We are =
currently running out of ideas.

>>=20
>> King regards
>> Andr=C3=A9
>>=20
>> --
>> Dr. Andr=C3=A9 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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