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Thank you all very much for the extremely useful replies!  The 405 nm
laser-produced ghost image was indeed the result of a misaligned
pinhole lens, especially the one for the 20X multi-immersion
objective, although all others (for 40X oil, 63X oil and 63X water)
had to be re-aligned as well. The system is now good as new.
  Thanks again; this is indeed a wonderful forum!

Zoltan


On 3/12/08, S. Pagakis (IIBEAA) <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello Zoltan
>
> we had a similar problem and was fixed by pinhole lens alignment. So
> this should do it for you.
> However, since sequential  +405 imaging was mentioned in this thread, I
> would like to report a problem we discovered on an SP5 recently. (LAS
> AF version 1.8.2)
>
> When someone is doing "by frame" or "by stack" sequential imaging, the
> software allows the selection of a different hardware settings per scan
> method.  This is OK so far.  However it also allows the selection of a
> different 405 pinhole lens per scan method, even if the objective lens
> is not changed between scans.
>
> Therefore it is possible to have a "wrong" 405 pinhole lens when a
> "non--405" scan has been defined.  Obviously this does not affect the
> images, because the wrong 405 pinhole lens is introduced when a
> different colour is acquired.
>
> The first problem this creates is a long delay switching between scan
> because it, unnecessarily, switches the 405 pinhole lens between scans.
>
> The BIGGEST problem however is when someone switches to "Line by line"
> sequential imaging, while a scan method with the wrong 405 pinhole lens
> is active.
>
> Then, because during "Line by Line' sequential scanning hardware
> changes are NOT allowed, the wrong 405-pinhole lens is used for ALL
> scan methods, even for the one which collects the 405 sugnal.  Then we
> have serious 405 image misalignment.
>
> It is, therefore, also possible that you are seeing these double images
> not because of misalignment of your corresponding pinhole lens but
> because the wrong 405-pinhole lens is used with the objective, EVEN IF
> YOU SELECTED THE CORRECT ONE WHEN STARTING THE EXPERIMENT.
>
> It is obviously a software "logic" bug and we will report it to Leica
> as well.
>
> Regards
>
>
> *********************************
> Stamatis Pagakis Ph.D.
> Biological Imaging Unit
> Biomedical Research Foundation, Academy of Athens
> Soranou Efessiou 4, Athens 115 27 - Greece
> M: 	+306946644955 				W: 	+302106597481
> FAX: +302106597545             		[log in to unmask]
>
>
> On 11 Mar 2008, at 03:09, Zoltan Cseresnyes wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > Dear All,
> >
> > I very much appreciate the replies I've received so far!!  The problem
> > may indeed be caused by a misaligned UV pinhole lens, which seems even
> > likelier in light of today's tests where I examined the same sample
> > with 4 different objectives (our system has separate UV pinhole lenses
> > for the 20x, 40x and 63x objectives, and no lens for the 10x).  The
> > results showed that the 10x objective produced ghost images throughout
> > the entire sample, whereas the 40x and 63x objectives produced no
> > ghost image at all.  I have a Leica engineer come in on Wednesday to
> > check and possibly re-align the system.  This will also give us a
> > chance to look inside for loose reflective items.
> >   Thanks very much again,
> >
> > 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