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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:[log in to unmask]] On Behalf Of Arvydas Matiukas
Sent: Wednesday, February 08, 2012 11:35 AM
To: [log in to unmask]
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.: 315-464-7997
fax: 315-464-8014
email: [log in to unmask]