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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:
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| 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.: 315-464-7997
| fax: 315-464-8014
| email: [log in to unmask]
|


-- 
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.