poor dapi images on LSM 510

[Boswell, Carl A - (cboswell)]

We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't make
things right.  We have had the AOM replaced once but the problem eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Julian Smith III]

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus FV1000; laser replaced under warranty).  New 405 is running at 5% on the same preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't make
things right.  We have had the AOM replaced once but the problem eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Guy Cox]

I can't speak for your particular problem, but there are problems with using a diode laser and an AOTF.  Diode lasers typically have a bit of wavelength spread, so with the narrow cut-off of an
AOTF you are likely to lose some power.  Diodes can also drift in wavelength - not usually enough to matter with a dichroic, but enough to bother an AOTF.  I suggest you check your 405 laser with a power meter and spectrum analyser to see what it is actually delivering.

                                              Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, III, Julian P.S
Sent: Saturday, 28 February 2009 10:57 AM
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus FV1000; laser replaced under warranty).  New 405 is running at 5% on the same preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality seems to decay over time (months) and pinhole/collimator tuning doesn't make things right.  We have had the AOM replaced once but the problem eventually returned.  We now have to use 100% 405 laser, rather than 10% to get anything close to good images.  Is there something about the AOM stability in this light path, or alignment issues with the laser that others have found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

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[O'Toole, P]

You may also want to check that the optic fibre is not being knocked. The
405 output on the 510 is sensitive to the optic fibre being moved close to
the back of the laser module.  This could account for the decay over time if
the laser output remains high.

Best

Pete

----- Original Message -----
From: "Carl Boswell" <[hidden email]>
To: <[hidden email]>
Sent: Friday, February 27, 2009 11:19 PM
Subject: poor dapi images on LSM 510

> We have been having a chronic problem with the image quality (S/N,
> resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
> seems to decay over time (months) and pinhole/collimator tuning doesn't
> make things right.  We have had the AOM replaced once but the problem
> eventually returned.  We now have to use 100% 405 laser, rather than 10%
> to get anything close to good images.  Is there something about the AOM
> stability in this light path, or alignment issues with the laser that
> others have found to result in a slow decay in usefulness of this channel?
>
> Thanks for your thoughts.
> Carl
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]

[Keith Morris]

Yep we had the same problem with our DAPI laser last year [3years old]. Kept
adjusting the collimator as illumination was uneven. DAPI faint etc..,
initially with duff samples, then with nuclei that dazzle under the mercury
lamp. Got the engineer out under the maintenance contract and he spent two
days trying to work out what was wrong [laser was reading very low]. Never
quite said what he thought it was. Came back and replaced the 405nm laser,
AOTF, and probably some related stuff [lot of boxes], and from then on the
405nm laser images have been fine [10% power]. The 405nm laser failure was
very gradual, so for a month or so didn't know quite what was going on.
Seemed very poor image quality was more noticeable than any lack of laser
power.

Had a related 405nm problem with the DAPI nuclei sometimes seeming to be in
a different Z plain to the FITC/TRITC z slice [could see a nuclei 'hole'
shadow but the DAPI was out of focus elsewhere. Seemed to come & go. I was
new to the post and the Zeiss 510, and hadn't noticed this sort of thing
before with other Leica/Bio-Rad confocals. Couldn't find a way to Z correct
the DAPI channel in LSM software [isn't one]. Had a chat with our Zeiss
confocal rep, and he said I expect you have a Plan Neofluar 40x and a Plan
Apochromat 63x [which we do]. The cheaper Plan Neofluar will cause this
problems apparently, while if you see it on the Plan Apochromat call in the
engineer. Hadn't twigged it was only being seen on the 40x, and probably
always had high power Apochromats before. So now we tend to look at the
nucleus and nucleoli with the 63x Apochromat.    

Keith  

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Smith, III, Julian P.S
Sent: 27 February 2009 23:57
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus FV1000;
laser replaced under warranty).  New 405 is running at 5% on the same preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't make

things right.  We have had the AOM replaced once but the problem eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[simon walker (BI)]

In my experience the major cause of loss of signal from 405 excitation
is the pinhole drifting out of alignment.  If it's significantly out of
alignment it can also cause the apparent large shift in focal plane you
describe.  This is a much more significant effect than the one you see
when you image eg. 1 um  beads and measure channel registration using
plan apos and neofluar objectives.
I know Carl said in his original posting that he had tried tuning the
pinhole, but I would always look at this first before worrying about
AOTFs etc.
Simon


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Keith Morris
Sent: 02 March 2009 10:10
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Yep we had the same problem with our DAPI laser last year [3years old].
Kept
adjusting the collimator as illumination was uneven. DAPI faint etc..,
initially with duff samples, then with nuclei that dazzle under the
mercury
lamp. Got the engineer out under the maintenance contract and he spent
two
days trying to work out what was wrong [laser was reading very low].
Never
quite said what he thought it was. Came back and replaced the 405nm
laser,
AOTF, and probably some related stuff [lot of boxes], and from then on
the
405nm laser images have been fine [10% power]. The 405nm laser failure
was
very gradual, so for a month or so didn't know quite what was going on.
Seemed very poor image quality was more noticeable than any lack of
laser
power.

Had a related 405nm problem with the DAPI nuclei sometimes seeming to be
in
a different Z plain to the FITC/TRITC z slice [could see a nuclei 'hole'
shadow but the DAPI was out of focus elsewhere. Seemed to come & go. I
was
new to the post and the Zeiss 510, and hadn't noticed this sort of thing
before with other Leica/Bio-Rad confocals. Couldn't find a way to Z
correct
the DAPI channel in LSM software [isn't one]. Had a chat with our Zeiss
confocal rep, and he said I expect you have a Plan Neofluar 40x and a
Plan
Apochromat 63x [which we do]. The cheaper Plan Neofluar will cause this
problems apparently, while if you see it on the Plan Apochromat call in
the
engineer. Hadn't twigged it was only being seen on the 40x, and probably
always had high power Apochromats before. So now we tend to look at the
nucleus and nucleoli with the 63x Apochromat.    

Keith  

------------------------------------------------------------------------
---
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On
Behalf Of Smith, III, Julian P.S
Sent: 27 February 2009 23:57
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus
FV1000;
laser replaced under warranty).  New 405 is running at 5% on the same
preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't
make

things right.  We have had the AOM replaced once but the problem
eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM
stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Rietdorf, Jens]

Similar problems over here, take a look at the beam profile of the
laser. It seems that can change if the laser ages. We had some success
with adjusting the fiber coupling at the laser coupling side, though we
have to do it from time to time.

Best, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of simon walker (BI)
Sent: Monday, March 02, 2009 11:38 AM
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

In my experience the major cause of loss of signal from 405 excitation
is the pinhole drifting out of alignment.  If it's significantly out of
alignment it can also cause the apparent large shift in focal plane you
describe.  This is a much more significant effect than the one you see
when you image eg. 1 um  beads and measure channel registration using
plan apos and neofluar objectives.
I know Carl said in his original posting that he had tried tuning the
pinhole, but I would always look at this first before worrying about
AOTFs etc.
Simon


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Keith Morris
Sent: 02 March 2009 10:10
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Yep we had the same problem with our DAPI laser last year [3years old].
Kept
adjusting the collimator as illumination was uneven. DAPI faint etc..,
initially with duff samples, then with nuclei that dazzle under the
mercury
lamp. Got the engineer out under the maintenance contract and he spent
two
days trying to work out what was wrong [laser was reading very low].
Never
quite said what he thought it was. Came back and replaced the 405nm
laser,
AOTF, and probably some related stuff [lot of boxes], and from then on
the
405nm laser images have been fine [10% power]. The 405nm laser failure
was
very gradual, so for a month or so didn't know quite what was going on.
Seemed very poor image quality was more noticeable than any lack of
laser
power.

Had a related 405nm problem with the DAPI nuclei sometimes seeming to be
in
a different Z plain to the FITC/TRITC z slice [could see a nuclei 'hole'
shadow but the DAPI was out of focus elsewhere. Seemed to come & go. I
was
new to the post and the Zeiss 510, and hadn't noticed this sort of thing
before with other Leica/Bio-Rad confocals. Couldn't find a way to Z
correct
the DAPI channel in LSM software [isn't one]. Had a chat with our Zeiss
confocal rep, and he said I expect you have a Plan Neofluar 40x and a
Plan
Apochromat 63x [which we do]. The cheaper Plan Neofluar will cause this
problems apparently, while if you see it on the Plan Apochromat call in
the
engineer. Hadn't twigged it was only being seen on the 40x, and probably
always had high power Apochromats before. So now we tend to look at the
nucleus and nucleoli with the 63x Apochromat.    

Keith  

------------------------------------------------------------------------
---
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On
Behalf Of Smith, III, Julian P.S
Sent: 27 February 2009 23:57
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus
FV1000;
laser replaced under warranty).  New 405 is running at 5% on the same
preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't
make

things right.  We have had the AOM replaced once but the problem
eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM
stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Keith Morris]

I notice the new Zeiss 710 Meta only has one pinhole, for pretty much this
reason I suspect. With our failing 405nm laser & associated AOTF optics
[whatever it was that was failing], pinhole adjustment made no difference
whatsoever [we do that regularly anyway]. We did find we were adjusting the
collimator a heck of a lot [despite the engineer saying you rarely have to
adjust it]. In the end the DAPI signal became more fuzz and noise than
signal, and increasing laser power made no difference [just brighter fuzz] -
by then the engineer was already involved. Even the Zeiss engineer took two
or three stabs to fix it [first time he thought it was OK, we didn't].  

Keith

---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of simon walker (BI)
Sent: 02 March 2009 10:38
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

In my experience the major cause of loss of signal from 405 excitation
is the pinhole drifting out of alignment.  If it's significantly out of
alignment it can also cause the apparent large shift in focal plane you
describe.  This is a much more significant effect than the one you see
when you image eg. 1 um  beads and measure channel registration using
plan apos and neofluar objectives.
I know Carl said in his original posting that he had tried tuning the
pinhole, but I would always look at this first before worrying about
AOTFs etc.
Simon


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Keith Morris
Sent: 02 March 2009 10:10
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Yep we had the same problem with our DAPI laser last year [3years old].
Kept
adjusting the collimator as illumination was uneven. DAPI faint etc..,
initially with duff samples, then with nuclei that dazzle under the
mercury
lamp. Got the engineer out under the maintenance contract and he spent
two
days trying to work out what was wrong [laser was reading very low].
Never
quite said what he thought it was. Came back and replaced the 405nm
laser,
AOTF, and probably some related stuff [lot of boxes], and from then on
the
405nm laser images have been fine [10% power]. The 405nm laser failure
was
very gradual, so for a month or so didn't know quite what was going on.
Seemed very poor image quality was more noticeable than any lack of
laser
power.

Had a related 405nm problem with the DAPI nuclei sometimes seeming to be
in
a different Z plain to the FITC/TRITC z slice [could see a nuclei 'hole'
shadow but the DAPI was out of focus elsewhere. Seemed to come & go. I
was
new to the post and the Zeiss 510, and hadn't noticed this sort of thing
before with other Leica/Bio-Rad confocals. Couldn't find a way to Z
correct
the DAPI channel in LSM software [isn't one]. Had a chat with our Zeiss
confocal rep, and he said I expect you have a Plan Neofluar 40x and a
Plan
Apochromat 63x [which we do]. The cheaper Plan Neofluar will cause this
problems apparently, while if you see it on the Plan Apochromat call in
the
engineer. Hadn't twigged it was only being seen on the 40x, and probably
always had high power Apochromats before. So now we tend to look at the
nucleus and nucleoli with the 63x Apochromat.    

Keith  

------------------------------------------------------------------------
---
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford  OX3 7BN,
United Kingdom.

Telephone:  [phone]
Email:  [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On
Behalf Of Smith, III, Julian P.S
Sent: 27 February 2009 23:57
To: [hidden email]
Subject: Re: poor dapi images on LSM 510

Sudden death of the 405?  We lost ours at 560+/- user hours (Olympus
FV1000;
laser replaced under warranty).  New 405 is running at 5% on the same
preps.
JSIII

-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [hidden email]
Subject: poor dapi images on LSM 510
 
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta.  The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't
make

things right.  We have had the AOM replaced once but the problem
eventually
returned.  We now have to use 100% 405 laser, rather than 10% to get
anything close to good images.  Is there something about the AOM
stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?

Thanks for your thoughts.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[MICROSCOPIA CONFOCAL y CCD]

Good afternoon. I am planning to use QUASAR 670,
from Biosearch Technologies, for peptide
labeling. I chose this molecule because its
spectral properties and prize. I would like to
know if anyone has experience with this
fluorophore. Is it a quite photo-stable
compound?, would you recommend it as a far-red
fluorophore?. Does anyone know problems regarding this compound?.
Thank you very much for your time.


At 14:31 02/03/2009, you wrote:
>[hidden email]

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Fernando González Camacho
[hidden email]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32