http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581978.html
Thanks for your post. We have thought about the Mie scattering. It will
pronounced 'distortion'. In Ch.11 of Pawley's Handbook of Confocal Micro.,
lenses including phase information. There I think they were also using gold
beads as sample. So I was wondering if my case is due to some other things,
The webapp is really helpful. Thanks for that again.
8 St. Mary St., Boston, MA, 02215
> From: Confocal Microscopy List [mailto:
>
[hidden email]] On Behalf Of Yan, Lu
> Sent: vendredi 2 mai 2014 03:24
> To:
[hidden email]
> Subject: Re: PSF measurement using Au beads
>
> On May 1, 2014 6:35 PM, "MODEL, MICHAEL" <
[hidden email]>
> wrote:
>
> From: Confocal Microscopy List <
>
[hidden email]> on
> behalf of Lu <
[hidden email]>
> Sent: Thursday, May 01, 2014 5:49 PM
> To:
[hidden email]
> Subject: PSF measurement using Au beads
>
> 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?
>
> 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.
>
> 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 probe the focal intensity distribution which somewhat
> related to the PSF of the system, so I just figured it might be easier to
> measure in this way, and i wanted to know if they had similar
> problems. But this distorted PSF seems to be related to the fact that the
> incident beam has non perpect Gaussian profile, which confuses me most.
>
> Hi Lu,
>
> I think that Mike is right about having to consider the angular dependence
> of scattering from the beads. (I'm assuming that your beam is collimated at
> the back focal plane). In confocal setups you illuminate your bead with a
> number of plane waves (i.e. k-vectors) that span a solid cone. Each plane
> wave within this cone is going to independently scatter light into a
> pattern that you can determine using Mie theory. Because the scattering is
> coherent, the total scattered field is just the sum of the different field
> scattering profiles from all the plane waves traveling in different
> directions within the cone, and the intensity that you measure is a
> projection of the squared-field distribution that fits within your system's
> numerical aperture onto a plane.
>
> All that being said, if you reduce the size of your beam in the back focal
> plane of the objective, then this is equivalent to illuminating your beads
> with a cone of light with a smaller apex angle. Equivalently, you have
> fewer plane waves that scatter and the total scattered field is summed over
> a smaller number of Mie field profiles. I suspect that this reduces any
> interference effects in the total scattered field and is what eliminates
> the lobes you're observing.
>
> To summarize, the messy axial profile might not originate from a dirty
> excitation beam, but simply because you're exciting the sphere with a
> number of plane waves traveling in different directions and the scattered
> fields from each of these plane waves is are interfering. If the spheres
> are touching the glass, this could also introduce asymmetries in the
> profile but I doubt it since you mentioned the spheres are mounted in
> glycerol, which has a refractive index close to glass.
>
> You could check the Mie profiles with this handy webapp:
>
http://omlc.ogi.edu/cgi-bin/mie_angles.cgi?diameter=0.15&lambda_vac=0.650&n_medium=1.47&nr_sphere=0.18&ni_sphere=-3.42&n_angles=100&density=0.1>
> I already entered the material parameters based on what you mentioned, but
> you should double check them anyway.
>
> Good luck!
>
> Kyle
>
> Dr. Kyle M. Douglass
> Postdoctoral Fellow
> The Laboratory of Experimental Biophysics
> EPFL, Lausanne, Switzerland
> +41 21 69 30556 (Office)
>
>
>