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Hi Andrea

is this drift always in the same direction?  We have observed (J.
Microsc. 210(2):131-137) significant focus drift on our Leica SP, linked
to temperature fluctuations in the room, but it was of an oscillatory
nature.  Could it be that the microscope stand keeps getting warmer with time?

Another thing to check is whether the stage has become loose and drifts
on the dovetail.

Hope it helps

***
Dr Stamatis Pagakis
Confocal microscopy and Image Analysis Laboratory
National Institute for Medical Research
The Ridgeway, Mill Hill, London NW7 1AA
Tel: +44 (0)20 8816 2621
Fax:  020 8906 4477
Mob: 07900 988 150

andrea manazza wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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

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