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Dear Mary,
 
Have you tried a thinner layer of Matrigel and image the spheroids with inverted microscopy? I presume you are using an upright. I don't know if this will work, but if you can gain access to an inverted system it may be worth trying.
 
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville, Victoria,      3050
Australia
Tel: 61 3 9341 3155    Fax: 61 3 9341 3104
email: [log in to unmask]
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________________________________

From: Confocal Microscopy List on behalf of Mary Olive
Sent: Tue 17/07/2007 1:08 AM
To: [log in to unmask]
Subject: Shadows in 3D cultures



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Hello, all,

I'm working with some people who are imaging (on an LSM 510) deep cultures
of tumor cell spheroids in Matrigel/DQ Collagen IV.  They are interested in
the spheroids, which are at the top of the culture, as well as the area
below.  Even when using a two-photon laser, the tumor cell spheroids seem to
cast a shadow, so that anything below them is not imaged.  A 3D
reconstruction shows a blank area from the bottom of the spheroid to the
bottom of the culture.  This 'blankness' is very visible when there is high
background from the DQ Collagen.

Is there anything that can be done to correct this?

Thanks in advance,

________________
Mary B. Olive
Microscopy & Imaging Resources Laboratory
Department of Pharmacology
Wayne State University, School of Medicine
Detroit, MI

313-577-2511