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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 <[log in to unmask]>:

> *****
> 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]
>



Jeremy Adler
IGP
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