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Good morning to all of you.
I've just started a few weeks ago performing time-lapses on my TCS SP2 
scope and I had to face a nasty trouble: I'm working on COS or NIH3T3 
cells, transfected with a chimeric GFP protein supposed to cooperate 
with actin during adhesion/migration processes. I'd like to see its 
movements during both processes.
I've been suggested by a more experienced colleague, who works fine on 
an old SP1, to keep the detection pinhole opened, put the objective in 
the vey middle of the dish -supposing you have cells there- and keep 
the laser as low as possible.
Unlikely, after no more than 20', I start loosing the cells: they seem 
to disappear, ruffles before and - from the outskirts inside - even the 
nucleus: I then decided to move the objective down, some 2 microns/10', 
and then I could keep the cells visible. I thought it could be due to 
the adhesion process, but it kept on going even after 8 hours...quite 
odd isn't it?
I know it might be quite trivial, but I'm not as experienced as most of 
you.
Any suggestion and criticism would be gratefully accepted, both for the 
loss of focus trouble and for some other related, useful tricks.
Thank you in advance, and have a nice time.
Andrew

dott. Andrea Manazza
Dipartimento di Oncologia, Università di Torino
CeRMS, ospedale Molinette
Via Santena, 7 - 10126 Torino, Italia
tel: +39-11-6706522
lab: +39-11-6336859
fax: +39-11-6336887