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horizontal dark lines appearing

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Aryeh Weiss

horizontal dark lines appearing

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We see (apparently) periodic horizontal dark lines with our Leica SP5,
when using the argon ion laser. Images that show these lines can be seen
at the following link:
http://cl.ly/2m2h0R1u1J2w

There were some some posts on the list about this, with the suggestion
that the laser is dying. However, when last I checked (a few weeks ago),
that laser was putting out plenty of power (although the 488nm line was
relatively weak compared to the 514nm line). Also, the lines have some
periodicity (50Hz?). Faster scan rates increase the line spacing, but it
does not appear to be linear with scan speed.
Frame averaging will average it out, so it is not correlated with the
scanning system. So the lines are annoying, but average signal seems fine.

None of the other lasers do this, and it is independent of detector.
Therefore, we assume that the problem is upstream of the vis laser
combiner/AOTF. That leaves the fiber, laser head, and power supply.

We suspect the laser, but it would be more satisfying to understand what
causes this behavior. Understanding the cause may lead to knowing how to
fix it without replacing the laser, but even if not, I like to know why
things work (or dont work) the way they do.

Has anyone has seen something similar?

Thanks in advance
--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

Nynne Meyn Christensen

Objective inverter

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Dear list,

We are thinking of buying a objective inverter (http://www.lsmtech.com/product_objective_inverter.html) to our Leica SP5X does anyone have any recommendations? Good and bad experiences then we would be happy to hear about it.

Thanks!

Best wishes,
Nynne

Nynne Meyn Christensen
Center Manager
Center for Advanced Bioimaging (CAB)
Department of Biology
University of Copenhagen
August Krogh, 6th floor, room 636
Universitetsparken 13
2200 København N

Guy Cox-2

Re: horizontal dark lines appearing

In reply to this post by Aryeh Weiss

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If the laser isn't dying, I don't know. But the common cause is that with a failing laser the feedback mechanism tries to bring it up to power, but this takes the current up to its limit so it trips out, and then the cycle starts again. I've seen it many times .... Since the 488 line is weak, that's what I'd suspect. But as the 514 is still strong this suggests that just realigning the laser will probably be enough to cure the problem.

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Aryeh Weiss
Sent: Friday, 31 August 2012 10:36 PM
To: [hidden email]
Subject: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We see (apparently) periodic horizontal dark lines with our Leica SP5, when using the argon ion laser. Images that show these lines can be seen at the following link:
http://cl.ly/2m2h0R1u1J2w

There were some some posts on the list about this, with the suggestion that the laser is dying. However, when last I checked (a few weeks ago), that laser was putting out plenty of power (although the 488nm line was relatively weak compared to the 514nm line). Also, the lines have some periodicity (50Hz?). Faster scan rates increase the line spacing, but it does not appear to be linear with scan speed.
Frame averaging will average it out, so it is not correlated with the scanning system. So the lines are annoying, but average signal seems fine.

None of the other lasers do this, and it is independent of detector.
Therefore, we assume that the problem is upstream of the vis laser combiner/AOTF. That leaves the fiber, laser head, and power supply.

We suspect the laser, but it would be more satisfying to understand what causes this behavior. Understanding the cause may lead to knowing how to fix it without replacing the laser, but even if not, I like to know why things work (or dont work) the way they do.

Has anyone has seen something similar?

Thanks in advance
--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

Farid Jalali

Re: horizontal dark lines appearing

In reply to this post by Aryeh Weiss

Hello Aryeh and Listers,
I've seen this several times with argon lasers and there are at least 2 reasons I found which can cause this issue. First, repeated fast live scans, where the live scans are at 1/2 or 1/4 of the horizontal spatial resolution and perhaps too much laser power or the preview is going on for too long. Going to the final scan at full spatial resolution reveals the stripes which are likely just bleach lines. Move to a new field and if there are no stripes, I think this explanation may hold up. Second, if the argon laser is slightly older, and has a rheostat on it for tuning power, I have found adjusting this can alleviate the banding you see. I do not know the exact explanation for why the banding results when this power tuning is off, but I do know that I have been able to alleviate the issue by tuning the rheostat. There may be other reasons like Guy's suggestion, these are just what I have experience with. Good luck. Cheers
Farid.
Sent on the TELUS Mobility network with BlackBerry

-----Original Message-----
From: Aryeh Weiss <[hidden email]>
Sender: Confocal Microscopy List <[hidden email]>
Date: Fri, 31 Aug 2012 15:35:44
To: <[hidden email]>
Reply-To: [hidden email]
Subject: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We see (apparently) periodic horizontal dark lines with our Leica SP5,
when using the argon ion laser. Images that show these lines can be seen
at the following link:
http://cl.ly/2m2h0R1u1J2w

There were some some posts on the list about this, with the suggestion
that the laser is dying. However, when last I checked (a few weeks ago),
that laser was putting out plenty of power (although the 488nm line was
relatively weak compared to the 514nm line). Also, the lines have some
periodicity (50Hz?). Faster scan rates increase the line spacing, but it
does not appear to be linear with scan speed.
Frame averaging will average it out, so it is not correlated with the
scanning system. So the lines are annoying, but average signal seems fine.

None of the other lasers do this, and it is independent of detector.
Therefore, we assume that the problem is upstream of the vis laser
combiner/AOTF. That leaves the fiber, laser head, and power supply.

We suspect the laser, but it would be more satisfying to understand what
causes this behavior. Understanding the cause may lead to knowing how to
fix it without replacing the laser, but even if not, I like to know why
things work (or dont work) the way they do.

Has anyone has seen something similar?

Thanks in advance
--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

Craig Brideau

Re: Objective inverter

In reply to this post by Nynne Meyn Christensen

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I would confirm with the company that it is achromatic over the wavelengths
you plan to use. For instance some optical systems are not well corrected
for the violet ranges, so if you are working in that range confirm that the
inverter relay tube is achromatic for those wavelengths. If you are
working in the normal cyan through red you should be fine as these are
quite standard.

Craig

On Fri, Aug 31, 2012 at 7:24 AM, Nynne Meyn Christensen <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> We are thinking of buying a objective inverter (
> http://www.lsmtech.com/product_objective_inverter.html) to our Leica SP5X
> does anyone have any recommendations? Good and bad experiences then we
> would be happy to hear about it.
>
> Thanks!
>
> Best wishes,
> Nynne
>
>
> Nynne Meyn Christensen
> Center Manager
> Center for Advanced Bioimaging (CAB)
> Department of Biology
> University of Copenhagen
> August Krogh, 6th floor, room 636
> Universitetsparken 13
> 2200 København N
>

Arvydas Matiukas

Re: horizontal dark lines appearing

In reply to this post by Aryeh Weiss

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Hello Aryeh,

I recently have been seeing black (actually only darker, as in your
images intensity does not drop to zero as well) lines or bands
in Ar laser 488nm line excited scans on our LSM510. It turned
out that one of the fans in the VIS laser unit was failing. The unit
houses all visual lasers, AOTFs with control electronics, and beam
combiner. Replacing the failing fan fixed the problem which likely
was related to the overheating of laser power supplies and/or AOTF
control electronics. This was very quick and cheap fix (after
somewhat prolonged diagnostics) .The additional oberved characteristic feature was that
the black lines started appearing after 1 hr after the start of cold
machine, and progressively have been yielding more fluctuation.

I am not well familiar with Leica confocals, and your tiff images
do not provide complete hardware settings so I cannot translate
my LSM510 diagnosis to your SP5. However, based on common confocal
machine principles I would suggest to check the temperature/cooling
regime and stability for your confocal. In my case lines were not
periodic but maybe overheating leads to some periodic oscillations
in SP5 electronics (or laser as Guy suggested earlier).

By the way take a plastic fluorescent slide (which is uniform and not easy to bleach)
and check if the dark lines still appear periodically. Starting with simple
test objects sometimes helps the diagnostics.

Good luck,
Arvydas
*******************

Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
Department of Pharmacology
SUNY Upstate Medical University
766 Irving Ave., WH 3167
Syracuse, NY 13210
tel.: 315-464-7997
fax: 315-464-8014
email: [hidden email]

>>> Aryeh Weiss <[hidden email]> 8/31/2012 8:35 AM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We see (apparently) periodic horizontal dark lines with our Leica SP5,
when using the argon ion laser. Images that show these lines can be seen
at the following link:
http://cl.ly/2m2h0R1u1J2w

There were some some posts on the list about this, with the suggestion
that the laser is dying. However, when last I checked (a few weeks ago),
that laser was putting out plenty of power (although the 488nm line was
relatively weak compared to the 514nm line). Also, the lines have some
periodicity (50Hz?). Faster scan rates increase the line spacing, but it
does not appear to be linear with scan speed.
Frame averaging will average it out, so it is not correlated with the
scanning system. So the lines are annoying, but average signal seems fine.

None of the other lasers do this, and it is independent of detector.
Therefore, we assume that the problem is upstream of the vis laser
combiner/AOTF. That leaves the fiber, laser head, and power supply.

We suspect the laser, but it would be more satisfying to understand what
causes this behavior. Understanding the cause may lead to knowing how to
fix it without replacing the laser, but even if not, I like to know why
things work (or dont work) the way they do.

Has anyone has seen something similar?

Thanks in advance
--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

Aryeh Weiss

Re: horizontal dark lines appearing

In reply to this post by Farid Jalali

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

Thank you for many list members who responded on and off-list.

I can summarize the responses as follows:

1. Some suspect the AOTF and/or the AOBS. Although other lasers do not
exhibit the problem, these devices are tuned for each line. However, the
fact that this problem occurs for each of the 4 argon lines, and not for
561 , 594 and 633, makes me doubt it is the AOTF.

2. Some suggest thermal issues. I checked the fans, and they all work
(if they did not, that laser would shut down pretty fast). However, I
cannot rule out a faulty thermal sensor, and am contacting Lasos to see
how this can be checked.

3. I ran some scans with a fluorescent plastic slide. The results are
uploaded to http://cl.ly/3K1L1S321925 . This is a LIF file, so you need
a LIF reader for it. ImageJ will do, but it is easier to see the
metadata with LAS Lite. Seems hard to find, but this link seems ok:
http://cifweb.unil.ch/index.php?option=com_docman&task=doc_details&gid=26&Itemid=57
This time I included the transmitted light images, which show the same
effect. I think I can rule out photobleaching. There are various
combinations of channels, simultaneous, and sequential scanning, using
488nm or 514nm (argon ion laser) and 561nm (DPSS laser).

I again thank everyone who responded, and will update on the list as I
obtain more information.

--aryeh
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

>
> -----Original Message-----
> From: Aryeh Weiss <[hidden email]>
> Sender: Confocal Microscopy List <[hidden email]>
> Date: Fri, 31 Aug 2012 15:35:44
> To: <[hidden email]>
> Reply-To: [hidden email]
> Subject: horizontal dark lines appearing
>
>
> We see (apparently) periodic horizontal dark lines with our Leica SP5,
> when using the argon ion laser. Images that show these lines can be seen
> at the following link:
> http://cl.ly/2m2h0R1u1J2w
>
> There were some some posts on the list about this, with the suggestion
> that the laser is dying. However, when last I checked (a few weeks ago),
> that laser was putting out plenty of power (although the 488nm line was
> relatively weak compared to the 514nm line). Also, the lines have some
> periodicity (50Hz?). Faster scan rates increase the line spacing, but it
> does not appear to be linear with scan speed.
> Frame averaging will average it out, so it is not correlated with the
> scanning system. So the lines are annoying, but average signal seems fine.
>
> None of the other lasers do this, and it is independent of detector.
> Therefore, we assume that the problem is upstream of the vis laser
> combiner/AOTF. That leaves the fiber, laser head, and power supply.
>
> We suspect the laser, but it would be more satisfying to understand what
> causes this behavior. Understanding the cause may lead to knowing how to
> fix it without replacing the laser, but even if not, I like to know why
> things work (or dont work) the way they do.
>
> Has anyone has seen something similar?
>
> Thanks in advance
> --aryeh
>

JOEL B. SHEFFIELD

Fwd: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

---------- Forwarded message ----------
From: JOEL B. SHEFFIELD <[hidden email]>
Date: Sun, Sep 2, 2012 at 10:46 AM
Subject: Re: horizontal dark lines appearing
To: [hidden email]

Hi Aryeh,

We have a Leica SP1 with an argon laser, and we too see these horizontal
lines at times, mostly with transmitted DIC images. In general, we remove
them with the FFT routines in ImageJ. Our Leica tech suggested that the
problem is that the polarization in the laser "wobbles" a bit, so that in
concert with the other polarized elements of the optical path, such as the
Wolleston filters, we get these horizontal patterns. Have you tried
removing all polarizing filters from the optical path?

Joel

On Sun, Sep 2, 2012 at 9:34 AM, Aryeh Weiss <[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>
> *****
>
> Thank you for many list members who responded on and off-list.
>
> I can summarize the responses as follows:
>
> 1. Some suspect the AOTF and/or the AOBS. Although other lasers do not
> exhibit the problem, these devices are tuned for each line. However, the
> fact that this problem occurs for each of the 4 argon lines, and not for
> 561 , 594 and 633, makes me doubt it is the AOTF.
>
> 2. Some suggest thermal issues. I checked the fans, and they all work (if
> they did not, that laser would shut down pretty fast). However, I cannot
> rule out a faulty thermal sensor, and am contacting Lasos to see how this
> can be checked.
>
> 3. I ran some scans with a fluorescent plastic slide. The results are
> uploaded to http://cl.ly/3K1L1S321925 . This is a LIF file, so you need a
> LIF reader for it. ImageJ will do, but it is easier to see the metadata
> with LAS Lite. Seems hard to find, but this link seems ok:
> http://cifweb.unil.ch/index.**php?option=com_docman&task=**
> doc_details&gid=26&Itemid=57<http://cifweb.unil.ch/index.php?option=com_docman&task=doc_details&gid=26&Itemid=57>
> This time I included the transmitted light images, which show the same
> effect. I think I can rule out photobleaching. There are various
> combinations of channels, simultaneous, and sequential scanning, using
> 488nm or 514nm (argon ion laser) and 561nm (DPSS laser).
>
> I again thank everyone who responded, and will update on the list as I
> obtain more information.
>
> --aryeh
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph: 972-3-5317638
> FAX: 972-3-7384051
>
>
>
>> -----Original Message-----
>> From: Aryeh Weiss <[hidden email]>
>> Sender: Confocal Microscopy List <[hidden email].**EDU<[hidden email]>
>> >
>> Date: Fri, 31 Aug 2012 15:35:44
>> To: <[hidden email].**EDU<[hidden email]>
>> >
>> Reply-To: [hidden email]
>> Subject: horizontal dark lines appearing
>>
>>
>> We see (apparently) periodic horizontal dark lines with our Leica SP5,
>> when using the argon ion laser. Images that show these lines can be seen
>> at the following link:
>> http://cl.ly/2m2h0R1u1J2w
>>
>> There were some some posts on the list about this, with the suggestion
>> that the laser is dying. However, when last I checked (a few weeks ago),
>> that laser was putting out plenty of power (although the 488nm line was
>> relatively weak compared to the 514nm line). Also, the lines have some
>> periodicity (50Hz?). Faster scan rates increase the line spacing, but it
>> does not appear to be linear with scan speed.
>> Frame averaging will average it out, so it is not correlated with the
>> scanning system. So the lines are annoying, but average signal seems fine.
>>
>> None of the other lasers do this, and it is independent of detector.
>> Therefore, we assume that the problem is upstream of the vis laser
>> combiner/AOTF. That leaves the fiber, laser head, and power supply.
>>
>> We suspect the laser, but it would be more satisfying to understand what
>> causes this behavior. Understanding the cause may lead to knowing how to
>> fix it without replacing the laser, but even if not, I like to know why
>> things work (or dont work) the way they do.
>>
>> Has anyone has seen something similar?
>>
>> Thanks in advance
>> --aryeh
>>
>>

Elizabeth Nickless

Re: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

HI

WE get horizontal lines with our zeiss LSM 510 when using transmission mode mostly. They increase/change frequency at faster scanning speeds and higher laser energy. We were told by our tech that it is stray light getting into the system. We have had to put blackout behind the existing black blinds we had across windows in our lab when doing transmission scans sometimes the light from the touch panel on the side of the zeiss is enough light but we turn it off if we are having real issues. WE don't have an issue if we do this and don't put the laser power/transmission up too high.

Liz Nickless

Microscopy and Imaging Centre
Senior Technical Officer
Analytical Development Section
Fonterra Innovations
Palmerston North
New Zealand
06 350 4649 x64527

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of JOEL B. SHEFFIELD
Sent: Monday, 3 September 2012 2:56 a.m.
To: [hidden email]
Subject: Fwd: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

---------- Forwarded message ----------
From: JOEL B. SHEFFIELD <[hidden email]>
Date: Sun, Sep 2, 2012 at 10:46 AM
Subject: Re: horizontal dark lines appearing
To: [hidden email]

Hi Aryeh,

We have a Leica SP1 with an argon laser, and we too see these horizontal
lines at times, mostly with transmitted DIC images. In general, we remove
them with the FFT routines in ImageJ. Our Leica tech suggested that the
problem is that the polarization in the laser "wobbles" a bit, so that in
concert with the other polarized elements of the optical path, such as the
Wolleston filters, we get these horizontal patterns. Have you tried
removing all polarizing filters from the optical path?

Joel

On Sun, Sep 2, 2012 at 9:34 AM, Aryeh Weiss <[hidden email]> wrote:

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

DISCLAIMER
This email contains information that is confidential and which may be legally privileged. If you have received this email in error, please notify the sender immediately and delete the email. This email is intended solely for the use of the intended recipient and you may not use or disclose this email in any way.

Aryeh Weiss

Re: horizontal dark lines appearing

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Liz and thank you for your response.

In our case, I can eliminate causes which would be common to all of the
lasers, since the problem only occurs with the argon-ion laser.
In the SP5, the vis lasers (all of them except the 405nm laser, which
for marketing reasons is referred to as "UV", when it is in fact "V")
are combined in the laser platform, and routed though the AOTF, and from
there via a fiber to the scanhead. There is no place (that I can see)
where a light leak can affect the argon ion laser independent of the
other vis lasers, since the laser platform is sealed.

--aryeh

On 9/2/12 11:26 PM, Elizabeth Nickless wrote:
> HI
>
> WE get horizontal lines with our zeiss LSM 510 when using
> transmission
mode mostly. They increase/change frequency at faster scanning speeds
and higher laser energy. We were told by our tech that it is stray light
getting into the system. We have had to put blackout behind the existing
black blinds we had across windows in our lab when doing transmission
scans sometimes the light from the touch panel on the side of the zeiss
is enough light but we turn it off if we are having real issues. WE
don't have an issue if we do this and don't put the laser
power/transmission up too high.

> Liz Nickless
>
> Microscopy and Imaging Centre
> Senior Technical Officer
> Analytical Development Section
> Fonterra Innovations
> Palmerston North
> New Zealand
> 06 350 4649 x64527
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of JOEL B. SHEFFIELD
> Sent: Monday, 3 September 2012 2:56 a.m.
> To: [hidden email]
> Subject: Fwd: horizontal dark lines appearing
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> ---------- Forwarded message ----------
> From: JOEL B. SHEFFIELD <[hidden email]>
> Date: Sun, Sep 2, 2012 at 10:46 AM
> Subject: Re: horizontal dark lines appearing
> To: [hidden email]
>
>
> Hi Aryeh,
>
> We have a Leica SP1 with an argon laser, and we too see these horizontal
> lines at times, mostly with transmitted DIC images. In general, we remove
> them with the FFT routines in ImageJ. Our Leica tech suggested that the
> problem is that the polarization in the laser "wobbles" a bit, so that in
> concert with the other polarized elements of the optical path, such as the
> Wolleston filters, we get these horizontal patterns. Have you tried
> removing all polarizing filters from the optical path?
>
> Joel
>
>
>
> On Sun, Sep 2, 2012 at 9:34 AM, Aryeh Weiss <[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>
>> *****
>>
>> Thank you for many list members who responded on and off-list.
>>
>> I can summarize the responses as follows:
>>
>> 1. Some suspect the AOTF and/or the AOBS. Although other lasers do not
>> exhibit the problem, these devices are tuned for each line. However, the
>> fact that this problem occurs for each of the 4 argon lines, and not for
>> 561 , 594 and 633, makes me doubt it is the AOTF.
>>
>> 2. Some suggest thermal issues. I checked the fans, and they all work (if
>> they did not, that laser would shut down pretty fast). However, I cannot
>> rule out a faulty thermal sensor, and am contacting Lasos to see how this
>> can be checked.
>>
>> 3. I ran some scans with a fluorescent plastic slide. The results are
>> uploaded to http://cl.ly/3K1L1S321925 . This is a LIF file, so you need a
>> LIF reader for it. ImageJ will do, but it is easier to see the metadata
>> with LAS Lite. Seems hard to find, but this link seems ok:
>> http://cifweb.unil.ch/index.**php?option=com_docman&task=**
>> doc_details&gid=26&Itemid=57<http://cifweb.unil.ch/index.php?option=com_docman&task=doc_details&gid=26&Itemid=57>
>> This time I included the transmitted light images, which show the same
>> effect. I think I can rule out photobleaching. There are various
>> combinations of channels, simultaneous, and sequential scanning, using
>> 488nm or 514nm (argon ion laser) and 561nm (DPSS laser).
>>
>> I again thank everyone who responded, and will update on the list as I
>> obtain more information.
>>
>> --aryeh
>>
>>> -----Original Message-----
>>> From: Aryeh Weiss <[hidden email]>
>>> Sender: Confocal Microscopy List <[hidden email].**EDU<[hidden email]>
>>>>
>>> Date: Fri, 31 Aug 2012 15:35:44
>>> To: <[hidden email].**EDU<[hidden email]>
>>>>
>>> Reply-To: [hidden email]
>>> Subject: horizontal dark lines appearing
>>>
>>>
>>> We see (apparently) periodic horizontal dark lines with our Leica SP5,
>>> when using the argon ion laser. Images that show these lines can be seen
>>> at the following link:
>>> http://cl.ly/2m2h0R1u1J2w
>>>
>>> There were some some posts on the list about this, with the suggestion
>>> that the laser is dying. However, when last I checked (a few weeks ago),
>>> that laser was putting out plenty of power (although the 488nm line was
>>> relatively weak compared to the 514nm line). Also, the lines have some
>>> periodicity (50Hz?). Faster scan rates increase the line spacing, but it
>>> does not appear to be linear with scan speed.
>>> Frame averaging will average it out, so it is not correlated with the
>>> scanning system. So the lines are annoying, but average signal seems fine.
>>>
>>> None of the other lasers do this, and it is independent of detector.
>>> Therefore, we assume that the problem is upstream of the vis laser
>>> combiner/AOTF. That leaves the fiber, laser head, and power supply.
>>>
>>> We suspect the laser, but it would be more satisfying to understand what
>>> causes this behavior. Understanding the cause may lead to knowing how to
>>> fix it without replacing the laser, but even if not, I like to know why
>>> things work (or dont work) the way they do.
>>>
>>> Has anyone has seen something similar?
>>>
>>> Thanks in advance
>>> --aryeh
>>>
>>>
>
>
> --
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384051

Alessandro Esposito

Re: horizontal dark lines appearing

In reply to this post by Aryeh Weiss

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Dear Aryeh,
I've experienced three causes of horizontal bands on a Leica SP5.

1.) Issues of initialization of the electronics resulting in horizontal bands. This
is very simple do diagnose and it is not what you see.
2.) 50-100Hz banding caused by light in the room (monitors or light bulbs).
Again, this is not what you see.
3.) Horizontal lines that are photobleached into the sample. This has been an
issue on our system and seen with both a Ti:Sapphire laser and a Ar:ion. What
really puzzled me is that I see the bands on screen only when I change pixel
resolution or field of view rotation. For nice fixed and mounted samples, it is
possible to see the lines also through the eyepiece. The issue can be
characterized using fluorescent plastic and high power settings of the laser. It
is a while I am not checking the issue, but in the past I had the impression
that happened only with one objective. Furthermore, the photobleached
regions were much darker than what was actually imaged by the confocal,
giving the impression that the problem was during the re-tracing of the laser,
perhaps happening when the pixel resolution was much lower of the diffraction
limit with the blanking of the laser not working at all. I got this from another
reply to this post. However, I must say we tested the blanking of the lasers
and was working. We changed both AOM and EOM controllers. If I will find the
time I will re-check the issue and post a follow-up.

Cheers,

Alessandro