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Stephan,
I am surprised to read that beads still move in 5% agarose. We do mount beads in 1.5% low-melting agarose for PSF measurements and dual-channel alignment on a regular base and have no problems with movements. For an upright system with water dipping lenses I would try a two-layer approach. First coat a plastic dish with a layer of agarose without beads, let it solidify, and put a second layer of agarose with beads on top. This would avoid beads accumulating at the agarose-plastic interface. Make sure to keep the second layer thin, to avoid touching/pressing the agarose with your objective while trying to find the beads. Based on our experience I would also recommend to not go beyond 2% agarose concentration since this decreases optical quality, apparently due to the change in refractive index. If you cover the agarose with the same medium you used for preparing the agarose, shrinking should not be an issue either. And the agarose will not dry.
Good luck,
Michael
On Jun 13, 2012, at 6:35 PM, Stephan Junek wrote:
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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
>
> [log in to unmask]
> T: +49 69 506820 – 2008
> F: +49 69 506820 – 2002
_____________
Michael Weber
PhD Student, Huisken lab
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108, 01307 Dresden
Tel. 0049351/2102837
http://www.mpi-cbg.de/research/research-groups/jan-huisken
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