Glicerin labeling

[jlribas]

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Dear all,
For one special experiment of one of my users, it would be very
interesting to label glicerin molecules with some fluorescent molecule.
Does anyone know if there is something in the market?
Thank you very much in advance

Best regards
Juan Luis

--
Juan Luis Ribas
Servicio de Microscopía
Centro de Investigación, Tecnología e Innovación
Universidad de Sevilla
Av. Reina Mercedes 4b
41012 Sevilla

Tfno: 954559983

[Arvydas Matiukas]

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Dear list,
 
Please advice what is the most efficient/cheap way to deal
with confocal laser instability. Our LSM 510 is 7 years old
but  Ar and Hene 543 lasers have been replaced two years
ago and have been used no more that 2000-2500 hrs.
Users started complaining about 488nm and 543nm laser
line stability, and I measured it using Chroma fluorescent
plastic slides and 10x objective. The frame- averaged fluorescent
signal is reduced by 40-50% in 1hr after laser start. The decline
curve is quite steady after first 3-5 min with some fluctuations.
 
The  most disturbing  are intermittent large scale signal fluctuations (2-4 fold)
occurring on about a minute timescale. They kill dynamic (e.g. FRET) or
long-term imaging. They happen irregularly, on average once in 4-5 days,
and always after 2-3 hrs after the lasers are started.
 
In a related post several years ago laser cooling, laser polarization, AOTF cooling,
and AOTF driver stability were listed as the key factors. I am quite confident that cooling
is not an issue as the fans within the equipment boxes are working well, and whole room
has a powerful local conditioning system which maintains ambient temperature very stable.
Zeiss engineer checked the fibers recently, and suggested that maybe AOTF drivers are
not stable. As we are not on service contract and have to pay for every replacement I
would be interested to do more testing myself to identify the failing component.
 
Any suggestions/advices are very welcome. I can provide the measurement curves if
somebody is intersted (contact me off list).
 
Thanks,
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.: [phone]
fax: [phone]
email: [hidden email]

[Unruh, Jay]

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If Zeiss will let you (and if you are laser safety trained), I would get a good power meter and measure the power over time directly at the laser output.  Then do the same between the AOTF output and the fiber (make sure the second order beam is blocked) and then at the objective back aperture (with point scan selected and a long time lapse with long pixel dwell time selected).  That will tell you where the problem is.  If the lasers are the problem, they are probably close to death.  I've only seen that kind of fluctuation when the Ar ion tube gets to really low pressure.  Given the fact that you see the same problem with the HeNe, it is probably an AOTF issue (you could check if the HeNe and Ar ion fluctuations are correlated with multitrack imaging).  I don't know much about the AOTF instability, but your zeiss service person could probably tell you whether it is the actual crystal or the driver.  Fiber issues are unlikely to cause rapid fluctuations unless something is vibrating the fiber and the mount is loose or it has a break in it.  Usually in those cases, the power is way down as well.  Also there shouldn't be a correlation with start time for the fiber.  You could try just gently moving the fiber during imaging and see if there are big changes.

Good Luck!
Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Arvydas Matiukas
Sent: Wednesday, February 08, 2012 11:35 AM
To: [hidden email]
Subject: laser stability

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Dear list,
 
Please advice what is the most efficient/cheap way to deal with confocal laser instability. Our LSM 510 is 7 years old but  Ar and Hene 543 lasers have been replaced two years ago and have been used no more that 2000-2500 hrs.
Users started complaining about 488nm and 543nm laser line stability, and I measured it using Chroma fluorescent plastic slides and 10x objective. The frame- averaged fluorescent signal is reduced by 40-50% in 1hr after laser start. The decline curve is quite steady after first 3-5 min with some fluctuations.
 
The  most disturbing  are intermittent large scale signal fluctuations (2-4 fold) occurring on about a minute timescale. They kill dynamic (e.g. FRET) or long-term imaging. They happen irregularly, on average once in 4-5 days, and always after 2-3 hrs after the lasers are started.
 
In a related post several years ago laser cooling, laser polarization, AOTF cooling, and AOTF driver stability were listed as the key factors. I am quite confident that cooling is not an issue as the fans within the equipment boxes are working well, and whole room has a powerful local conditioning system which maintains ambient temperature very stable.
Zeiss engineer checked the fibers recently, and suggested that maybe AOTF drivers are not stable. As we are not on service contract and have to pay for every replacement I would be interested to do more testing myself to identify the failing component.
 
Any suggestions/advices are very welcome. I can provide the measurement curves if somebody is intersted (contact me off list).
 
Thanks,
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.: [phone]
fax: [phone]
email: [hidden email]

[Jeremy Adler-4]

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If you cannot prevent the fluctuations in laser power you can attempt  
to correct for them.
Zeiss used to have a diode to detect laser power (our one never worked  
properly), which will record an image that records the laser power -  
use this image to correct ( by division) the fluorescence images.
Similarly, depending in the sample, you can collect a transmitted  
light image while collecting the fluorescence. This will record the  
laser power and can also be used as a correction.

For the correction the laser power image and the fluorescent image  
need to have zero intensity set correctly. Then divide the fluorescent  
image with the laser intensity and rescale by the mean laser  
intensity. Not perfect and assumes a linear relationship between power  
and fluorescence, but will make a big improvement.




Quoting Arvydas Matiukas <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Please advice what is the most efficient/cheap way to deal
> with confocal laser instability. Our LSM 510 is 7 years old
> but  Ar and Hene 543 lasers have been replaced two years
> ago and have been used no more that 2000-2500 hrs.
> Users started complaining about 488nm and 543nm laser
> line stability, and I measured it using Chroma fluorescent
> plastic slides and 10x objective. The frame- averaged fluorescent
> signal is reduced by 40-50% in 1hr after laser start. The decline
> curve is quite steady after first 3-5 min with some fluctuations.
>
> The  most disturbing  are intermittent large scale signal  
> fluctuations (2-4 fold)
> occurring on about a minute timescale. They kill dynamic (e.g. FRET) or
> long-term imaging. They happen irregularly, on average once in 4-5 days,
> and always after 2-3 hrs after the lasers are started.
>
> In a related post several years ago laser cooling, laser  
> polarization, AOTF cooling,
> and AOTF driver stability were listed as the key factors. I am quite  
> confident that cooling
> is not an issue as the fans within the equipment boxes are working  
> well, and whole room
> has a powerful local conditioning system which maintains ambient  
> temperature very stable.
> Zeiss engineer checked the fibers recently, and suggested that maybe  
> AOTF drivers are
> not stable. As we are not on service contract and have to pay for  
> every replacement I
> would be interested to do more testing myself to identify the  
> failing component.
>
> Any suggestions/advices are very welcome. I can provide the  
> measurement curves if
> somebody is intersted (contact me off list).
>
> Thanks,
> 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.: [phone]
> fax: [phone]
> email: [hidden email]
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[George McNamara]

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Hi Arvydas,

Highly reminiscent of the LSM510 I manage(d) here in Miami (PC died
recently - finally able to decommission it - if you or anyone else needs
parts, make UM an offer ... I am putting on a digital camera system on
the Axiovert 200M stand, though only may be able to use the right side
camera port - left side may have some factory installed features
specific for the LSM light path).

It took Zeiss a lot longer than I would have liked, but one of their
field service engineer's greatly improved out LSM510 by removing the ~25
mm of dust that had assumulated in the dust trap under the electronics box.


See this paper for the nice, the bad and the ugly of other confocal's:

             Quality assurance testing for modern optical imaging
systems. </pubmed/21477410> Stack RF, Bayles CJ, Girard AM, Martin K,
Opansky C, Schulz K, *Cole RW*. Microsc Microanal. 2011
Aug;17(4):598-606. Epub 2011 Apr 11. PMID: 21477410


In other news ... our Leica SP5 inverted microscope had a very dim PMT3.
This week, Leica field service (thanks Jim C and Gene B) replaced the
PMT - it is now the brightest. They will be visiting in a few weeks to
replace three other PMTs in that SP5, plus the 3 standard PMTs in our
MP/SP5/FCS/FLIM - having a service contract is a good thing. Each
machine is close to 5 years old and have been sustaining close to 1,000
hours of use (Sp5 inverted use now up thanks to retiring the old LSM510).

George


On 2/8/2012 12:34 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Please advice what is the most efficient/cheap way to deal
> with confocal laser instability. Our LSM 510 is 7 years old
> but  Ar and Hene 543 lasers have been replaced two years
> ago and have been used no more that 2000-2500 hrs.
> Users started complaining about 488nm and 543nm laser
> line stability, and I measured it using Chroma fluorescent
> plastic slides and 10x objective. The frame- averaged fluorescent
> signal is reduced by 40-50% in 1hr after laser start. The decline
> curve is quite steady after first 3-5 min with some fluctuations.
>
> The  most disturbing  are intermittent large scale signal fluctuations (2-4 fold)
> occurring on about a minute timescale. They kill dynamic (e.g. FRET) or
> long-term imaging. They happen irregularly, on average once in 4-5 days,
> and always after 2-3 hrs after the lasers are started.
>
> In a related post several years ago laser cooling, laser polarization, AOTF cooling,
> and AOTF driver stability were listed as the key factors. I am quite confident that cooling
> is not an issue as the fans within the equipment boxes are working well, and whole room
> has a powerful local conditioning system which maintains ambient temperature very stable.
> Zeiss engineer checked the fibers recently, and suggested that maybe AOTF drivers are
> not stable. As we are not on service contract and have to pay for every replacement I
> would be interested to do more testing myself to identify the failing component.
>
> Any suggestions/advices are very welcome. I can provide the measurement curves if
> somebody is intersted (contact me off list).
>
> Thanks,
> 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.: [phone]
> fax: [phone]
> email: [hidden email]
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[mcammer]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

A few weeks ago I posted a message that we were having strange fluctuations of intensity with our Zeiss 710 confocal.  Turns out the AOTF controller was shot.  Also, there was a question whether the "crystal" had gone bad.  Regardless, Zeiss service quickly fixed the problem and tuned up the system with more laser power than before it went down and it has been stable since (about 6 weeks so far).
Regards,
Michael
________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Thursday, February 09, 2012 3:25 AM
To: [hidden email]
Subject: Re: laser stability

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

If you cannot prevent the fluctuations in laser power you can attempt  
to correct for them.
Zeiss used to have a diode to detect laser power (our one never worked  
properly), which will record an image that records the laser power -  
use this image to correct ( by division) the fluorescence images.
Similarly, depending in the sample, you can collect a transmitted  
light image while collecting the fluorescence. This will record the  
laser power and can also be used as a correction.

For the correction the laser power image and the fluorescent image  
need to have zero intensity set correctly. Then divide the fluorescent  
image with the laser intensity and rescale by the mean laser  
intensity. Not perfect and assumes a linear relationship between power  
and fluorescence, but will make a big improvement.




Quoting Arvydas Matiukas <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Please advice what is the most efficient/cheap way to deal
> with confocal laser instability. Our LSM 510 is 7 years old
> but  Ar and Hene 543 lasers have been replaced two years
> ago and have been used no more that 2000-2500 hrs.
> Users started complaining about 488nm and 543nm laser
> line stability, and I measured it using Chroma fluorescent
> plastic slides and 10x objective. The frame- averaged fluorescent
> signal is reduced by 40-50% in 1hr after laser start. The decline
> curve is quite steady after first 3-5 min with some fluctuations.
>
> The  most disturbing  are intermittent large scale signal  
> fluctuations (2-4 fold)
> occurring on about a minute timescale. They kill dynamic (e.g. FRET) or
> long-term imaging. They happen irregularly, on average once in 4-5 days,
> and always after 2-3 hrs after the lasers are started.
>
> In a related post several years ago laser cooling, laser  
> polarization, AOTF cooling,
> and AOTF driver stability were listed as the key factors. I am quite  
> confident that cooling
> is not an issue as the fans within the equipment boxes are working  
> well, and whole room
> has a powerful local conditioning system which maintains ambient  
> temperature very stable.
> Zeiss engineer checked the fibers recently, and suggested that maybe  
> AOTF drivers are
> not stable. As we are not on service contract and have to pay for  
> every replacement I
> would be interested to do more testing myself to identify the  
> failing component.
>
> Any suggestions/advices are very welcome. I can provide the  
> measurement curves if
> somebody is intersted (contact me off list).
>
> Thanks,
> 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.: [phone]
> fax: [phone]
> email: [hidden email]
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

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[Peter Gabriel Pitrone]

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Hello Arvydas,

I did a Technician Qualification called the Technology of the Microscope
through the Royal Microscopical Society (TechRMS) on this subject. I
submit to you my thesis, which might be able to help you find your problem
or at least a method to help find it (I hope the link works, if not write
me back privately):

http://mpi-cbg.academia.edu/PeterPitrone/Books/1240206/METHODS_FOR_DETERMINING_THE_STABILITY_AND_RELIABILITY_OF_THE_ILLUMINATION_SYSTEM_OF_A_LASER_SCANNING_CONFOCAL_MICROSCOPE

The little fan (~25mm^2) in the back of the AOTF controller box can get
warped by heat if your system isn't temperature stabilized, it's an easy
fix for about 5-10 bucks if you have a sure enough hand at that sort of
thing.

Good luck!!

Pete

On Wed, February 8, 2012 6:34 pm, Arvydas Matiukas wrote:
| *****
| To join, leave or search the confocal microscopy listserv, go to:
| http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
| *****
|
| Dear list,
|
| Please advice what is the most efficient/cheap way to deal
| with confocal laser instability. Our LSM 510 is 7 years old
| but  Ar and Hene 543 lasers have been replaced two years
| ago and have been used no more that 2000-2500 hrs.
| Users started complaining about 488nm and 543nm laser
| line stability, and I measured it using Chroma fluorescent
| plastic slides and 10x objective. The frame- averaged fluorescent
| signal is reduced by 40-50% in 1hr after laser start. The decline
| curve is quite steady after first 3-5 min with some fluctuations.
|
| The  most disturbing  are intermittent large scale signal fluctuations
| (2-4 fold)
| occurring on about a minute timescale. They kill dynamic (e.g. FRET) or
| long-term imaging. They happen irregularly, on average once in 4-5 days,
| and always after 2-3 hrs after the lasers are started.
|
| In a related post several years ago laser cooling, laser polarization,
| AOTF cooling,
| and AOTF driver stability were listed as the key factors. I am quite
| confident that cooling
| is not an issue as the fans within the equipment boxes are working well,
| and whole room
| has a powerful local conditioning system which maintains ambient
| temperature very stable.
| Zeiss engineer checked the fibers recently, and suggested that maybe AOTF
| drivers are
| not stable. As we are not on service contract and have to pay for every
| replacement I
| would be interested to do more testing myself to identify the failing
| component.
|
| Any suggestions/advices are very welcome. I can provide the measurement
| curves if
| somebody is intersted (contact me off list).
|
| Thanks,
| 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.: [phone]
| fax: [phone]
| email: [hidden email]
|


--
Peter Gabriel Pitrone - TechRMS
Microscopy/Imaging Specialist
Prof. Dr. Pavel Tomancak group
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden

"If a straight line fit is required, obtain only two data points." - Anon.