http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581964.html
Thanks for your reply. Yes you are right those two layers are cover glass
and glass slide. I have seen in many papers people using gold beads to
and i wanted to know if they had similar problems. But this distorted PSF
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> Hi Lu,
>
> It seems to me that reflecting beads would be a tricky object to get PSF
> from because you have to deal with the angular dependence of scattering and
> reflection. 8 um might be the distance between a slide and a coverslip,
> both surfaces should reflect.
>
> Mike Model
>
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> Subject: PSF measurement using Au beads
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> Hello all,
>
> We have a home-build confocal microscopy setup in our lab, and we have been
> trying to evaluate the PSF using gold beads (d=150 nm), but currently we
> are
> having some difficulty on interpreting some of our results. I am hopeful
> that I could find some help here.
>
> Backgroud: Our excitation light is a 650 nm laser diode. Objective lens is
> Olympus 60X 1.35/Oil, and the illumination light from a single mode fiber
> is
> collimated using a Thorlabs achromatic doublet with focal length of 30 mm,
> and then sent into the objective by folding mirrors (the beam is slightly
> underfilling the back aperture of the objective). Piezo scanning is used
> instead of resonant mirrors scanning. We collect the reflected/scattered
> light from the bead to form image. No filter was used in front of our
> detector. For the beads sample, 99% Glycerol was used as mountant.
>
> Problems:
> 1. Two reflective layers showed up as we do axially scanning, separated by
> about 8 um, and the bead turned out to be attached to one of them. The
> axial
> PSF looks terribly distorted. It is very much like a four lobes pattern,
> i.e. an intensity null surrounded by 4 lobes on top/bottom and right/left.
> You could imagine that at some z positions, the lateral intensity pattern
> has a donut-shape. We do have a good explanation why this happened. Does
> any
> one ever have similar problem? Is my sample preparation wrong?
>
> 2. If I put a iris before the back aperture of the objective, and closed it
> a little bit to truncated my collimated beam to half of its original size,
> then the axial PSF suddenly got cleaned up, i.e. a single nice vertical
> lobe
> appeared. But 2 reflective layers were still there observable. Any idea
> why?
> We thought the achromatic double for collimation might induce some higher
> order free space mode other than pure Gaussian mode, such that when we
> close
> the iris we effectively cut off some high k vectors of those 'other modes',
> leaving nicer Gaussian going into the objective to produce nicer axial PSF.
> Does this make sense to you guys?
>
> 3. A question often confuses me, which exactly quantity, in my case, should
> I correlate my measured FWHM of the bead image, in order to check if my
> setup is of diffraction limited performance? I have not been able to find a
> consistent criteria in literatures.
>
> Thanks in advance.
> Lu
>