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Hi Stephan,

> 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 did something like that recently, using 80 nm diameter nanogold
particles embedded in low-melt agarose and imaged in reflected light
mode on a Zeiss LSM 780 NLO with dip-in lenses. The advantage over using
a fluorescent bead is that the reflected signal is bright and of
narrowly defined spectral range (we wanted to define chromatic
aberration of our objectives, not get an experimental PSF for
deconvolution). Particle movement didn't seem to be too much of an
issue, perhaps because I could scan fast thanks to the high signal. Or
possibly, the agarose mixture was a bit different to yours. Or, it might
not have mattered for our question and I have forgotten. If I scrape my
neurons a bit harder I recall that the near-IR laser made the particles
wobble a bit, maybe because of local heating. Let me know if you want
details of the protocol.

Michael