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Are you imaging line by line, frame by frame , or stack by stack? We
had this same problem and our Leica rep (Linda Brown) suggested we do
either frame by frame or stack by stack scanning. This solved our
problem of what appeared to be bleed through but is really a very
energetic and long life time signal from the UV laser.
Chere Petty M.S.
Manager of UMBC Keith R. Porter Core Imaging Facility
Department of Biological Sciences
University of Maryland Baltimore County
(UMBC)
1000 Hilltop Circle
Baltimore Maryland 21250
301-367-8408
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emumbc.com
On Mar 10, 2008, at 9:19 AM, Zoltan Cseresnyes wrote:
> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Dear All,
>
> We have a fairly new Leica SP5 system on a DM6000 scope, equipped
> with AOBS/AOTF, 405 nm diode, argon, HeNe and 561 nm DPSS lasers.
> When imaging a monolayer of cells, in this case the epithelial cell
> layer of a fly embryo, using the 405 nm laser (Hoechst labelled) we
> get two in-focus images, appr. 10 microns apart. One of these is
> the "real" image, corresponding to the top of the embryo, whereas
> the second ("ghost") image appears deep inside the embryo. It is
> physically/biologically impossible to have a second layer of
> labelled epithelial cells inside the embryo, so it must be an
> optical effect. I tested the system with a double coverslip sample
> with a thin layer of air between the two coverslips (using two
> pieces of tape as spacers) in reflection mode, which revealed a
> very strong second maximum (optical intererence at the glass
> surface) with the 405 nm laser, but not with the other three
> lasers. The 405 nm lightpath avoids the AOBS, so I'm wondering if
> that holds the key to this problem. Leica is also trying to figure
> this out. Has anyone come across this weird behaviour?? Any help
> or advice will be much appreciated!
> Thanks,
>
> Zoltan
>
>
>
> --
> Zoltan Cseresnyes
> Facility manager, Imaging Suite
> Dept. of Zoology University of Cambridge
> Downing Street, Cambridge
> CB2 3EJ UK
>
> Tel.: (++44) (0)1223 769282
> Fax.: (++44) (0)1223 336676
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