Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Laser-stability-over-time-tp7587681p7587682.html
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Hi Milan,
It could be the specimen focus changes over time. This can -- and should
-- be evaluated by X-Z scan.
I recommend:
* continuing with your reflection slide, though consider sometimes
testing a fluorescent slide like Chroma's plastic slides (or a piece of
a fluorescent plastic clipboard). If you can afford it, ArgoLight's
calibration targets are supposed to be non-bleaching.
* imaging 50:50 edge of slide and air.
* turning on the transmitted light detector, operating that at modest
gain, helps to have a neutral density filter between the condenser
(which you can parfocalize by shrinking the condenser numerical aperture
to get it in focus) and detector.
* Each laser in its own scan track ... extremely low laser power (under
2% may be unstable because of the AOTF intensity control, not the lasers
themselves). I typically operate the lasers to the ChD transmitted image
is approximately the same brightness in all tracks (keep ChD gain constant).
* "no moving parts" (other than the scanners) in the light path.
* 1 minute interval overnight ... since now Friday, can do over the weekend.
* and sure, do this in X-Z scan (field rotated to accommodate edge).
* Do every night (room temperature and other variables may be different)
and over weekends.
Have low expectations!
I encourage you to post online your results and interpretation.
When i moved to UMiami in 2007 our LSM510 had huge fluctuations. One of
the Zeiss field service engineers found 6 (or more?) years of dust in
the trap under the RTC (real time computer), which was sitting on the
floor. After the dust trap was cleaned, the 510's fluctuations were
greatly diminished (good), though not eliminated (sigh). Too bad Zeiss
did not do thorough preventive maintenances every time (i.e. every 6
months while under warranty and service contract).
enjoy,
George
On 12/15/2017 5:34 AM, Milan Esner wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> Dear colleagues,
>
> we have new Zeiss LSM880 confocal microscope in our facility and performing measurements of laser stability over time.
> I am using slide with mirror and measuring reflection on internal detectors.
> However the results shows quite high fluctuation for all lasers.
> I would like to ask you if you are measuring the stability in time of lasers on your microscopes and what is the correct method for this measurement and also how big fluctuation you observe on your system?
>
> Thanks a lot for sharing your experience. Best regards,
>
> Milan
>
>
>
--
George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamarahttps://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650http://confocal.jhu.eduJuly 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstractsupporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures