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You could try either a higher agarose concentration, or think about using collagen (aka gelatin) or possibly even pluronic instead (because of its weird phase behaviour pluronic might be more resistant to local melting due to laser heating). I've successfully taken PSFs in an admittedly fairly concentrated (~ 30%) gelatin gel without any bead movement. The downside of gelatin is that it has some background fluorescence, but this is likely to be more of a problem in widefield. A second caveat is that a high concentration gel of either gelatin or agarose will have a refractive index higher than that of water. 

cheers,
David

________________________________
 From: Stephan Junek <[log in to unmask]>
To: [log in to unmask] 
Sent: Thursday, 14 June 2012 4:35 AM
Subject: Measuring PSFs using water dipping objective
 
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Dear all,

I am looking for a good protocol to measure PSFs on a confocal microscope
using water dipping objectives (objectives used for physiology, i.e. no
cover glass!). 
I tried using fluorescent beads embedded in 5 % low melting point agarose,
but it appears that the beads are moving, probably due to slight swelling of
the agarose which is immersed in the water.
I would prefer not to use a cover glass, as this probably introduces
aberrations. Using beads dried on a glass surface is not ideal either due to
the reflection at the water/glass interface.

Thanks in advance for any suggestions!

Best,
Stephan.


-- 
________________________________________
Dr. Stephan Junek
Max Planck Institute for Brain Research
Deutschordenstr. 46
60528 Frankfurt am Main
Germany

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