Similar problems over here, take a look at the beam profile of the
laser. It seems that can change if the laser ages. We had some success
with adjusting the fiber coupling at the laser coupling side, though we
have to do it from time to time.
Best, jens
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of simon walker (BI)
Sent: Monday, March 02, 2009 11:38 AM
To: [log in to unmask]
Subject: Re: poor dapi images on LSM 510
In my experience the major cause of loss of signal from 405 excitation
is the pinhole drifting out of alignment. If it's significantly out of
alignment it can also cause the apparent large shift in focal plane you
describe. This is a much more significant effect than the one you see
when you image eg. 1 um beads and measure channel registration using
plan apos and neofluar objectives.
I know Carl said in his original posting that he had tried tuning the
pinhole, but I would always look at this first before worrying about
AOTFs etc.
Simon
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Keith Morris
Sent: 02 March 2009 10:10
To: [log in to unmask]
Subject: Re: poor dapi images on LSM 510
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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