I can't speak for your particular problem, but there are problems with using a diode laser and an AOTF.  Diode lasers typically have a bit of wavelength spread, so with the narrow cut-off of an 
AOTF you are likely to lose some power.  Diodes can also drift in wavelength - not usually enough to matter with a dichroic, but enough to bother an AOTF.  I suggest you check your 405 laser with a power meter and spectrum analyser to see what it is actually delivering.

                                              Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Smith, III, Julian P.S
Sent: Saturday, 28 February 2009 10:57 AM
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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