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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
>
>
>
>
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