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This problem appears related to what I have down here: the info set in a
Method for the mixers (PMT, TLD, etc) seems to be saved in that method but,
when you select it, Lasersharp 3.2 frequently 'forgets' what it is supposed
to display (eg. Mixer will show at the heading: All at 0%, instead of PMT1
at 100% or TLD3 at 100%, etc...).
We either use Custom mode or edit the Method each time, which is a nuisance.
BioRad people gave it a try without a successful outcome to this bug.
Good luck,
Renato Mortara
-----Mensagem original-----
De: Confocal Microscopy List
[mailto:[log in to unmask]]Em nome de Martin Wessendorf
Enviada em: Terça-feira, 2 de Abril de 2002 13:13
Para: [log in to unmask]
Assunto: Photon-counting on Bio-Rad 1024
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Hey, folks--
We have a BioRad 1024 running LaserSharp 3.2 on an OS/2 machine. I've
recently tried to set up a "method" for imaging triple-labeled tissue
using photon-counting. I began by opening the default "triple-labeling"
method, going to "setup", pulling down to "method" and choosing
"save-as".
After that, I simply opened my new method and repeated the steps,
chosing "edit method", instead. When editing it, I expected that all
I'd need to do was to select the "mixer" button, choose photon-counting
("PhC" rather than "PMT"), and turn on the correct PMT for each mixer.
I did so, and was able to collect single images for each color.
However, the real trouble began when I attempted to collect a sequential
z-series. This is something we did all the time using the default
"triple-labeling" method, and so I was expecting no changes. The method
would appear to set everything up correctly, but when it came time to
collect the images, only one color would be imaged--for the other two it
would be as if the PMT was turned off: the image-stacks would be saved,
but would be empty, with every pixel at a value of zero. The color that
is successfully saved varies from occasion to occasion, which makes
things particularly confusing!
So...does anyone out there have any insight into what's going wrong?
Has anyone successfully made a method-file for photon-counting that
works?
Thanks!
Martin Wessendorf
--
Martin Wessendorf, Ph.D. office: (612) 626 0145
Assoc Prof, Dept Neuroscience lab: (612) 624 2991
University of Minnesota Preferred FAX: (612) 624 8118
6-145 Jackson Hall, 321 Church St. SE Dept FAX: (612) 626 5009
Minneapolis, MN 55455 e-mail: [log in to unmask]
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