Zeiss LSM 700 Confocal- laser problem

[Z.J. Zhang]

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

Hi everyone:

I am having a weird problem with my Zeiss LSM 700 confocal microscope and hope you could shed light on it -

When I tried to scan with the lasers off (turning the lasers off via the ZEN software), I can literally see a relatively bright light coming from the objective! The light looks white, which I assume is the combination of the blue, green and red lasers. Although the detected fluorescence signal is 'minimal', it still worries that 1). It might photobleach my sample; and 2). When I scan with one laser, the other lasers might be on as well, so I wouldn't know what exactly I am scanning with!

I was told by Zeiss that this is "normal" because there are no mechanical shutters in the scan head. I have a hard time to believe it and here are my questions:

1). Does anyone experience the same/similar problem?
2). What could be the possible cause?
3). How am I supposed to fix it?

Thank you,

Zhaojie

Zhaojie Zhang, Ph. D.
Director, Jenkins Microscopy Facility
University of Wyoming
Laramie, WY 82071
PHONE: [phone]
FAX: [phone]

[John Oreopoulos]

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

Zhaojie,

I'm not familiar with the LSM 700, but based on what you wrote where the Zeiss representatives said this is normal because their are no mechanical shutters, they could be referring to the fact that the confocal laser engine is AOTF-based, and therefore what you might be seeing is AOTF "leakage" of the laser light onto the specimen plane. I believe this is a common issue with AOTF-based laser engines. That is to say, the AOTF always passes a small (non-zero) amount of light into the diffracted order beam. Perhaps you can see it by eye because it is focused to a small spot at the front of the objective lens and it scatters from that point. Should you be concerned about leakage light reaching your sample? I think this is a valid question, but I'm not quite sure what the best test for that would be. I can think of a few.

Maybe other LSM 700 users can comment and confirm/deny this experience with their systems as well.

John Oreopoulos
Staff Scientist
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2013-08-12, at 1:11 PM, Z.J. Zhang wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone:
>
> I am having a weird problem with my Zeiss LSM 700 confocal microscope and hope you could shed light on it -
>
> When I tried to scan with the lasers off (turning the lasers off via the ZEN software), I can literally see a relatively bright light coming from the objective! The light looks white, which I assume is the combination of the blue, green and red lasers. Although the detected fluorescence signal is 'minimal', it still worries that 1). It might photobleach my sample; and 2). When I scan with one laser, the other lasers might be on as well, so I wouldn't know what exactly I am scanning with!
>
> I was told by Zeiss that this is "normal" because there are no mechanical shutters in the scan head. I have a hard time to believe it and here are my questions:
>
> 1). Does anyone experience the same/similar problem?
> 2). What could be the possible cause?
> 3). How am I supposed to fix it?
>
> Thank you,
>
> Zhaojie
>
> Zhaojie Zhang, Ph. D.
> Director, Jenkins Microscopy Facility
> University of Wyoming
> Laramie, WY 82071
> PHONE: [phone]
> FAX: [phone]

[Vladimir Zhukarev]

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

Hi John,
 
If your AOTF 'always passes a small (non-zero) amount of light into the diffracted order beam' but extensive enough, so that you can see the 'leakage' in the specimen plane, then your 'engine' is long time out of commission. And that is all to it.
 
Best
Vlad
--------- Original Message --------- Subject: Re: Zeiss LSM 700 Confocal- laser problem
From: "John Oreopoulos" <[hidden email]>
Date: 8/12/13 10:29 am
To: [hidden email]

*****
 To join, leave or search the confocal microscopy listserv, go to:
 http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
 *****
 
 Zhaojie,
 
 I'm not familiar with the LSM 700, but based on what you wrote where the Zeiss representatives said this is normal because their are no mechanical shutters, they could be referring to the fact that the confocal laser engine is AOTF-based, and therefore what you might be seeing is AOTF "leakage" of the laser light onto the specimen plane. I believe this is a common issue with AOTF-based laser engines. That is to say, the AOTF always passes a small (non-zero) amount of light into the diffracted order beam. Perhaps you can see it by eye because it is focused to a small spot at the front of the objective lens and it scatters from that point. Should you be concerned about leakage light reaching your sample? I think this is a valid question, but I'm not quite sure what the best test for that would be. I can think of a few.
 
 Maybe other LSM 700 users can comment and confirm/deny this experience with their systems as well.
 
 John Oreopoulos
 Staff Scientist
 Spectral Applied Research
 Richmond Hill, Ontario
 Canada
 www.spectral.ca
 
 
 On 2013-08-12, at 1:11 PM, Z.J. Zhang wrote:
 
 > *****
 > To join, leave or search the confocal microscopy listserv, go to:
 > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
 > *****
 >
 > Hi everyone:
 >
 > I am having a weird problem with my Zeiss LSM 700 confocal microscope and hope you could shed light on it -
 >
 > When I tried to scan with the lasers off (turning the lasers off via the ZEN software), I can literally see a relatively bright light coming from the objective! The light looks white, which I assume is the combination of the blue, green and red lasers. Although the detected fluorescence signal is 'minimal', it still worries that 1). It might photobleach my sample; and 2). When I scan with one laser, the other lasers might be on as well, so I wouldn't know what exactly I am scanning with!
 >
 > I was told by Zeiss that this is "normal" because there are no mechanical shutters in the scan head. I have a hard time to believe it and here are my questions:
 >
 > 1). Does anyone experience the same/similar problem?
 > 2). What could be the possible cause?
 > 3). How am I supposed to fix it?
 >
 > Thank you,
 >
 > Zhaojie
 >
 > Zhaojie Zhang, Ph. D.
 > Director, Jenkins Microscopy Facility
 > University of Wyoming
 > Laramie, WY 82071
 > PHONE: [phone]
 > FAX: [phone]

[G. Esteban Fernandez]

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

We've always seen that in our LSM 710 but the light is not from the lasers,
it is from the widefield excitation source (X-cite lamp).  We can close the
lamp's shutter to get rid of the stray light.  I've never seen it affect
the images but we close the shutter with dim specimens just to be safe.

-Esteban


On Mon, Aug 12, 2013 at 10:11 AM, Z.J. Zhang <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone:
>
> I am having a weird problem with my Zeiss LSM 700 confocal microscope and
> hope you could shed light on it -
>
> When I tried to scan with the lasers off (turning the lasers off via the
> ZEN software), I can literally see a relatively bright light coming from
> the objective! The light looks white, which I assume is the combination of
> the blue, green and red lasers. Although the detected fluorescence signal
> is 'minimal', it still worries that 1). It might photobleach my sample; and
> 2). When I scan with one laser, the other lasers might be on as well, so I
> wouldn't know what exactly I am scanning with!
>
> I was told by Zeiss that this is "normal" because there are no mechanical
> shutters in the scan head. I have a hard time to believe it and here are my
> questions:
>
> 1). Does anyone experience the same/similar problem?
> 2). What could be the possible cause?
> 3). How am I supposed to fix it?
>
> Thank you,
>
> Zhaojie
>
> Zhaojie Zhang, Ph. D.
> Director, Jenkins Microscopy Facility
> University of Wyoming
> Laramie, WY 82071
> PHONE: [phone]
> FAX: [phone]
>

[Edith Suss-Toby]

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

Does anybody have experience with exchanging the 510 Meta confocal lasers: 561nm or 405nm, not via Zeiss?
Is it possible? if yes where from can we purchase the lasers?
Thank you
Edith

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vladimir Zhukarev
Sent: Monday, August 12, 2013 11:39 PM
To: [hidden email]
Subject: Re: Zeiss LSM 700 Confocal- laser problem

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

Hi John,
 
If your AOTF 'always passes a small (non-zero) amount of light into the diffracted order beam' but extensive enough, so that you can see the 'leakage' in the specimen plane, then your 'engine' is long time out of commission. And that is all to it.
 
Best
Vlad
--------- Original Message --------- Subject: Re: Zeiss LSM 700 Confocal- laser problem
From: "John Oreopoulos" <[hidden email]>
Date: 8/12/13 10:29 am
To: [hidden email]

*****
 To join, leave or search the confocal microscopy listserv, go to:
 http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
 *****
 
 Zhaojie,
 
 I'm not familiar with the LSM 700, but based on what you wrote where the Zeiss representatives said this is normal because their are no mechanical shutters, they could be referring to the fact that the confocal laser engine is AOTF-based, and therefore what you might be seeing is AOTF "leakage" of the laser light onto the specimen plane. I believe this is a common issue with AOTF-based laser engines. That is to say, the AOTF always passes a small (non-zero) amount of light into the diffracted order beam. Perhaps you can see it by eye because it is focused to a small spot at the front of the objective lens and it scatters from that point. Should you be concerned about leakage light reaching your sample? I think this is a valid question, but I'm not quite sure what the best test for that would be. I can think of a few.
 
 Maybe other LSM 700 users can comment and confirm/deny this experience with their systems as well.
 
 John Oreopoulos
 Staff Scientist
 Spectral Applied Research
 Richmond Hill, Ontario
 Canada
 www.spectral.ca
 
 
 On 2013-08-12, at 1:11 PM, Z.J. Zhang wrote:
 
 > *****
 > To join, leave or search the confocal microscopy listserv, go to:
 > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
 > *****
 >
 > Hi everyone:
 >
 > I am having a weird problem with my Zeiss LSM 700 confocal microscope and hope you could shed light on it -  >  > When I tried to scan with the lasers off (turning the lasers off via the ZEN software), I can literally see a relatively bright light coming from the objective! The light looks white, which I assume is the combination of the blue, green and red lasers. Although the detected fluorescence signal is 'minimal', it still worries that 1). It might photobleach my sample; and 2). When I scan with one laser, the other lasers might be on as well, so I wouldn't know what exactly I am scanning with!
 >
 > I was told by Zeiss that this is "normal" because there are no mechanical shutters in the scan head. I have a hard time to believe it and here are my questions:
 >
 > 1). Does anyone experience the same/similar problem?
 > 2). What could be the possible cause?
 > 3). How am I supposed to fix it?
 >
 > Thank you,
 >
 > Zhaojie
 >
 > Zhaojie Zhang, Ph. D.
 > Director, Jenkins Microscopy Facility  > University of Wyoming  > Laramie, WY 82071  > PHONE: [phone]  > FAX: [phone]

[Pasierbek,Pawel]

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

Hi Zhaojie,

We had the same issue with our LSM700. The problem has been solved by our Zeiss engineer by tuning the "laser dark signal".  This solved the problem for us.
As far as I know there is no AOTF inside the lsm700 and lasers are directly modulated.

Hope this helps.

All the best,
Pawel
________________________________________
Von: Confocal Microscopy List [[hidden email]]&quot; im Auftrag von &quot;G. Esteban Fernandez [[hidden email]]
Gesendet: Dienstag, 13. August 2013 06:12
An: [hidden email]
Betreff: Re: Zeiss LSM 700 Confocal- laser problem

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

We've always seen that in our LSM 710 but the light is not from the lasers,
it is from the widefield excitation source (X-cite lamp).  We can close the
lamp's shutter to get rid of the stray light.  I've never seen it affect
the images but we close the shutter with dim specimens just to be safe.

-Esteban


On Mon, Aug 12, 2013 at 10:11 AM, Z.J. Zhang <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone:
>
> I am having a weird problem with my Zeiss LSM 700 confocal microscope and
> hope you could shed light on it -
>
> When I tried to scan with the lasers off (turning the lasers off via the
> ZEN software), I can literally see a relatively bright light coming from
> the objective! The light looks white, which I assume is the combination of
> the blue, green and red lasers. Although the detected fluorescence signal
> is 'minimal', it still worries that 1). It might photobleach my sample; and
> 2). When I scan with one laser, the other lasers might be on as well, so I
> wouldn't know what exactly I am scanning with!
>
> I was told by Zeiss that this is "normal" because there are no mechanical
> shutters in the scan head. I have a hard time to believe it and here are my
> questions:
>
> 1). Does anyone experience the same/similar problem?
> 2). What could be the possible cause?
> 3). How am I supposed to fix it?
>
> Thank you,
>
> Zhaojie
>
> Zhaojie Zhang, Ph. D.
> Director, Jenkins Microscopy Facility
> University of Wyoming
> Laramie, WY 82071
> PHONE: [phone]
> FAX: [phone]
>

[Jeremy Adler-4]

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

It is normal but irritating.

The lasers cannot be completely switched off - the LSM700 is a budget  
confocal.
However although you can see the laser light its level is very low -  
but with a bright specimen and pushing the detection limits it can be  
produce a detectabe image - scanning with the lasers nominally off.

There is therefore a risk of bleaching part of the specimen in a  
timeseries. A solution is to use the multiposition acquisition and  
sacrifice one position.
However we have not noticed a photobleaching problem - but it is easy to test.





Quoting "Z.J. Zhang" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone:
>
> I am having a weird problem with my Zeiss LSM 700 confocal  
> microscope and hope you could shed light on it -
>
> When I tried to scan with the lasers off (turning the lasers off via  
> the ZEN software), I can literally see a relatively bright light  
> coming from the objective! The light looks white, which I assume is  
> the combination of the blue, green and red lasers. Although the  
> detected fluorescence signal is 'minimal', it still worries that 1).  
> It might photobleach my sample; and 2). When I scan with one laser,  
> the other lasers might be on as well, so I wouldn't know what  
> exactly I am scanning with!
>
> I was told by Zeiss that this is "normal" because there are no  
> mechanical shutters in the scan head. I have a hard time to believe  
> it and here are my questions:
>
> 1). Does anyone experience the same/similar problem?
> 2). What could be the possible cause?
> 3). How am I supposed to fix it?
>
> Thank you,
>
> Zhaojie
>
> Zhaojie Zhang, Ph. D.
> Director, Jenkins Microscopy Facility
> University of Wyoming
> Laramie, WY 82071
> PHONE: [phone]
> FAX: [phone]
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[Pascal Weber]

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

I think you can do it yourself and even change the category of laser. And put
that DPSS but beware it will take you align yourself lasers to ensure their co-
alignment before entering the fiber. But you no longer have any warranty
available.

[lgelman]

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

Hi Zhang,

I agree with Pawel that this is the most probable explanation. It is known to happen with LSM700s, which indeed have no AOTF to keep their price low. A Zeiss technician can check/fix this very easily by changing laser-modulation parameters.

Best,

Laurent.
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Z.J. Zhang
Sent: 12 August 2013 19:11
To: [hidden email]
Subject: Zeiss LSM 700 Confocal- laser problem

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

Hi everyone:

I am having a weird problem with my Zeiss LSM 700 confocal microscope and hope you could shed light on it -

When I tried to scan with the lasers off (turning the lasers off via the ZEN software), I can literally see a relatively bright light coming from the objective! The light looks white, which I assume is the combination of the blue, green and red lasers. Although the detected fluorescence signal is 'minimal', it still worries that 1). It might photobleach my sample; and 2). When I scan with one laser, the other lasers might be on as well, so I wouldn't know what exactly I am scanning with!

I was told by Zeiss that this is "normal" because there are no mechanical shutters in the scan head. I have a hard time to believe it and here are my questions:

1). Does anyone experience the same/similar problem?
2). What could be the possible cause?
3). How am I supposed to fix it?

Thank you,

Zhaojie

Zhaojie Zhang, Ph. D.
Director, Jenkins Microscopy Facility
University of Wyoming
Laramie, WY 82071
PHONE: [phone]
FAX: [phone]