Yep we had the same problem with our DAPI laser last year [3years old]. Kept
adjusting the collimator as illumination was uneven. DAPI faint etc..,
initially with duff samples, then with nuclei that dazzle under the mercury
lamp. Got the engineer out under the maintenance contract and he spent two
days trying to work out what was wrong [laser was reading very low]. Never
quite said what he thought it was. Came back and replaced the 405nm laser,
AOTF, and probably some related stuff [lot of boxes], and from then on the
405nm laser images have been fine [10% power]. The 405nm laser failure was
very gradual, so for a month or so didn't know quite what was going on.
Seemed very poor image quality was more noticeable than any lack of laser
power.
Had a related 405nm problem with the DAPI nuclei sometimes seeming to be in
a different Z plain to the FITC/TRITC z slice [could see a nuclei 'hole'
shadow but the DAPI was out of focus elsewhere. Seemed to come & go. I was
new to the post and the Zeiss 510, and hadn't noticed this sort of thing
before with other Leica/Bio-Rad confocals. Couldn't find a way to Z correct
the DAPI channel in LSM software [isn't one]. Had a chat with our Zeiss
confocal rep, and he said I expect you have a Plan Neofluar 40x and a Plan
Apochromat 63x [which we do]. The cheaper Plan Neofluar will cause this
problems apparently, while if you see it on the Plan Apochromat call in the
engineer. Hadn't twigged it was only being seen on the 40x, and probably
always had high power Apochromats before. So now we tend to look at the
nucleus and nucleoli with the 63x Apochromat.
Keith
---------------------------------------------------------------------------
Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.
Telephone: +44 (0)1865 287568
Email: [log in to unmask]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Smith, III, Julian P.S
Sent: 27 February 2009 23:57
To: [log in to unmask]
Subject: Re: poor dapi images on LSM 510
Sudden death of the 405? We lost ours at 560+/- user hours (Olympus FV1000;
laser replaced under warranty). New 405 is running at 5% on the same preps.
JSIII
-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Fri 2/27/2009 6:19 PM
To: [log in to unmask]
Subject: poor dapi images on LSM 510
We have been having a chronic problem with the image quality (S/N,
resolution) of DAPI stained nuclei using a Zeiss 510 Meta. The quality
seems to decay over time (months) and pinhole/collimator tuning doesn't make
things right. We have had the AOM replaced once but the problem eventually
returned. We now have to use 100% 405 laser, rather than 10% to get
anything close to good images. Is there something about the AOM stability
in this light path, or alignment issues with the laser that others have
found to result in a slow decay in usefulness of this channel?
Thanks for your thoughts.
Carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
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