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Hi Kyle,
Thanks for your post. We have thought about the Mie scattering. It will
indeed affecting the measurement, but we did not expect to see that
pronounced 'distortion'. In Ch.11 of Pawley's Handbook of Confocal Micro.,
Juskaitis describes several methods to measure 'true' PSF of objective
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,
like I mentioned in the post that maybe the collimation lens does a so bad
job to create a Gaussian beam with nearly flat phase at around the back
aperture of my objective, such that a lot of other spatial modes also get
to enter into the objective to cause unexpected 'PSF'.
The webapp is really helpful. Thanks for that again.
Cheers,
Lu
-----------------------------------------------------
Lu Yan
Nanostructured Fibers and Nonlinear Optics Laboratory
Electrical and Computer Engineering
Boston University
8 St. Mary St., Boston, MA, 02215
(617)353-0286
[log in to unmask]
-----------------------------------------------------
On Fri, May 2, 2014 at 3:28 AM, Douglass Kyle Michael <[log in to unmask]
> wrote:
> From: Confocal Microscopy List [mailto:
> [log in to unmask]] On Behalf Of Yan, Lu
> Sent: vendredi 2 mai 2014 03:24
> To: [log in to unmask]
> Subject: Re: PSF measurement using Au beads
>
> On May 1, 2014 6:35 PM, "MODEL, MICHAEL" <[log in to unmask]>
> wrote:
>
> From: Confocal Microscopy List <
> [log in to unmask]> on
> behalf of Lu <[log in to unmask]>
> Sent: Thursday, May 01, 2014 5:49 PM
> To: [log in to unmask]
> 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)
>
>
>
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