Strange microscopy problem

[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

--
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

[Joao Lagarto]

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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

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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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[Andre Bernardini]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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 http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date: 10/12/15
>

[Steffen Dietzel]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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

[Csúcs Gábor-3]

Dear Andre,

Currently I do not have any good ideas, but questions:
1) Is this 10-15 cycle dependent on the number of positions (locations) or the number of images (imaging speed)?
2) Do you see this modulation also if the stage movement is so small that you virtually stay at the same field of view?
3) Do you have a temperature control system?

Greetings    Gabor


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andre Bernardini
Sent: Montag, 12. Oktober 2015 10:35
To: [hidden email]
Subject: Strange microscopy problem

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

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

[Eric Marino]

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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:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> Post images on http://www.imgur.com <http://www.imgur.com/> and include the link in your posting.
> *****
>
> 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:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> 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

[Joao Lagarto]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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 http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date:
> 10/12/15
>

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date: 10/12/15

[Andre Bernardini]

*****
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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy <http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> Post images on http://www.imgur.com <http://www.imgur.com/> and include the link in your posting.
>> *****
>>
>> 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:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> 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

[Peter O'Toole]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

We have seen a similar effect, on an incubated system with very similar timing and also not across all wavelengths. Our case was different as it was pinned down to an imperfect optic fibre for l;asers and the aircon heating/cooling the fibre itself. A change of fibre and shielding from room temperature effects fixed the problem. Other microscopes in the room have not exhibited the same problem. Therefore I would also try and discount any room aircon catching part of the light path and creating an intensity or optical shift.

Best
Pete

Dr Peter O'Toole
Head of Imaging and Cytometry
Technology Facility
Department of Biology (Area 15)
University of York
YORK
YO10 5DD

Tel : [phone]
email : [hidden email]
www.york.ac.uk/biology/tf
Twitter: @YorkBioimaging



EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joao Lagarto
Sent: 12 October 2015 15:29
To: [hidden email]
Subject: Re: Strange microscopy problem

*****
To join, leave or search the confocal microscopy listserv, go to:
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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

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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 http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date:
> 10/12/15
>

-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date: 10/12/15

[Nuno Moreno]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

Yes Peter

incubators have PID controllers and temperature is quite stable. HVAC systems vary as João was saying, with simple on/off control. Just try to switch the air conditioning off in the room and measure again.

Nuno Moreno
Scientific Facilities
Instituto Gulbenkian de Ciencia
 

> On 12 Oct 2015, at 15:39, Peter O'Toole <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We have seen a similar effect, on an incubated system with very similar timing and also not across all wavelengths. Our case was different as it was pinned down to an imperfect optic fibre for l;asers and the aircon heating/cooling the fibre itself. A change of fibre and shielding from room temperature effects fixed the problem. Other microscopes in the room have not exhibited the same problem. Therefore I would also try and discount any room aircon catching part of the light path and creating an intensity or optical shift.
>
> Best
> Pete
>
> Dr Peter O'Toole
> Head of Imaging and Cytometry
> Technology Facility
> Department of Biology (Area 15)
> University of York
> YORK
> YO10 5DD
>
> Tel : [phone]
> email : [hidden email]
> www.york.ac.uk/biology/tf
> Twitter: @YorkBioimaging
>
>
>
> EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joao Lagarto
> Sent: 12 October 2015 15:29
> To: [hidden email]
> Subject: Re: 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 http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> 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 http://www.imgur.com and include the link in your posting.
>> *****
>>
>> 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
>>
>>
>>
>> -----
>> No virus found in this message.
>> Checked by AVG - www.avg.com
>> Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date:
>> 10/12/15
>>
>
>
>
> -----
> No virus found in this message.
> Checked by AVG - www.avg.com
> Version: 2015.0.6172 / Virus Database: 4435/10804 - Release Date: 10/12/15

[Peter O'Toole]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

We are seeking maternity cover for one of our Senior Technicians in the
Imaging & Cytometry Laboratory within the Bioscience Technology
Facility, Department of Biology, University of York.

The Imaging & Cytometry Laboratory is one of the six, specialist core
laboratories within the Bioscience Technology Facility and focusses on
all aspects of confocal and electron microscopy, flow cytometry, plus
associated techniques.  It has an enviable international reputation for
training and instrument and method development.  The work of the
laboratory is wide and very varied and involves training, support and
advice; experimental troubleshooting and equipment maintenance; method
development. Our users are researchers from both the University of York
and external organisations (companies, other universities, Government
Institutes etc.).  Working closely with the Laboratory Head and the rest
of the team, you will be involved in delivering aspects of the work of
the Laboratory to our users.

This position represents an exciting opportunity to gain further
experience in the application of microscopy and flow cytometry to a very
wide range of challenging and interesting biological questions.  You
will be a team worker, have at least a degree in biology or related
discipline, and a good knowledge and experience of flow cytometry or
microscopy.

The post is available at 60% Full Time Equivalent from 4 November 2015
and is anticipated to be available until 9 August 2016 in the first
instance.

Best

Pete

--
Dr Peter O'Toole
Head of Imaging and Cytometry
Technology Facility
Department of Biology (Area 15)
University of York
YORK
YO10 5DD

Tel : [phone]
Fax : [phone]
email : [hidden email]
www.york.ac.uk/biology/tf

Times Higher Education University of the Year 2010

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