http://confocal-microscopy-list.275.s1.nabble.com/PSF-bead-shot-but-X-Y-and-Z-dont-match-tp4589883p4590412.html
What NA objective are you using? With an NA 1.4 oil immersion objective you should get a FWHM below 250nm. If you are getting 400nm then something is optically wrong - probably spherical aberration caused by a refractive index mismatch - things get pretty sensitive at this sort of NA. With a dry NA 0.75 NA objective you should do better than 450nm but only if the preparation is perfect - my advice is always to use an objective with a correction collar if you want to do serious quality imaging with a dry lens. (Yes, I know this is not a cheap option.)
The other thing to bear in mind is that 170nm isn't really small enough to count as a point object for an NA 1.4 objective - so there will be a convolution of the bead with the psf probably taking the image to larger than the true psf. I use 100nm or even 60nm beads to check with an oil lens - that way you can in practice ignore the contribution of the object.
I can't see much point in getting into deconvolution until your system is performing at its optical limit. (And even then I wouldn't expect too much).
Guy
Hey Fellow ListServers,
I purchased a PSF kit from Invitrogen and imaged the beads and when i
loaded the beads to correct the Z blur i measured the XY of the bead
to make sure the diameter matched but it turns out it doesnt. The
beads are supposedly .17um with +-.005 but whatever angle i measure
the bead at in xy i keep getting .4 um. Any ideas as to why this is
happening?
Thank You
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Matiar Jafari