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Hi Karl-Johan -
My expensive suggestion is to get a 63X water immersion lens and leave the cells in PBS or HBSS.
Here is some information comparing water and oil immersion lenses. http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr05/rvwimm.html
-Lars
Pfizer Global Research & Development
Ann Arbor Laboratories
2800 Plymouth Rd., Ann Arbor, MI 48105
Lars D. Engstrom
Senior Associate Scientist
Drug Safety Research & Development
Investigational Imaging & Cytometry
35/149-AF
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Ph: 734.622.7628
Fax: 734.622.3478
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Karl-Johan Leuchowius
Sent: Friday, November 17, 2006 6:23 AM
To: [log in to unmask]
Subject: Problems with confocal microscopy in 96 well plates
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Dear all,
I am trying to do multicolour fluorescent staining (Hoechst 33342, Alexa
488, Alexa 555, Texas Red and cy5) of cells grown in 96 well plates
(Nunc CytoWell with cover glass bottom) which I look at with a confocal
microscope (Zeiss 510 meta, 63x or 40x oil objective). I add Vectashield
to mount the cells and to protect the fluorophores from fading. This
procedure works nicely when I use cells grown on slides, mounted under a
cover glass.
The problem I'm experiencing with the staining in the 96 well plates is
that the vectashield seems to make everything look hazy--the staining
looks weaker and the background seems to increase. The more vectashield
I add, the worse it looks. Also, the haze effect grows stronger over
time, so that after a few hours all I can see is something that looks
like "fluorescent fog". I have also tried to do without the vectashield,
and the cells look great (bright and crisp staining, no background) when
looking through the lens. However, if I try to look at them confocally
they look terrible (probably because of optical effects).
Does anyone have any ideas of what to try? Would another mounting
medium, such as Prolong, work better? How much mounting medium should be
used?
Thanks for your help!
cheers,
Karl-Johan
--
Karl-Johan Leuchowius, M.Sc., PhD student
Molecular Medicine, Dept. of Genetics and Pathology
Uppsala University
Rudbeck Laboratory
Dag Hammarskjölds Väg 20
S-751 85 Uppsala
Sweden
Phone: +46 (0)18 471 4851
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