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But would this scattered photon really mess up the psf measurement?

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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]
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On Fri, May 2, 2014 at 4:42 PM, Craig Brideau <[log in to unmask]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Regarding the iris, when the iris is stopped down, you are reducing the NA
> of your objective. This will favour the collection of light from specular
> reflection, so the most ballistic photons coming off your sample will be
> admitted while any scatter will be rejected. This really only works for the
> case where you have a lot of specular reflection, such as with metal.
>
> Craig Brideau
>
>
> On Fri, May 2, 2014 at 4:15 AM, Zdenek Svindrych <[log in to unmask]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Lu,
> > 1. You can reduce the two reflective layers by closely matching the
> > refractive indices using proper oil instead of glycerol. The mismatch may
> > also influence the PSF itself, but not dramatically. Also by opening up
> the
> > detection pinhole you can scan the laser focus profile, this can tell you
> > whether the problem is due to excitation or detection.
> >
> > 2. 30 mm achromat doublet can introduce some aberrations. I often use low
> > power microscope objectives when it comes to short focal lengths. Also
> note
> > that to get the best diffraction-limited excitation you should
> > significantly
> > overfill the back aperture of the objective. So a 100 mm collimating lens
> > (achromat doublet) could solve both problems.
> >
> > Finally, try fluorescent beads. They should give you more accurate
> > representation of your PSF...
> >
> > Regards, zdenek svindrych
> >
> >
> >
> > ---------- Původní zpráva ----------
> > Od: Lu <[log in to unmask]>
> > Komu: [log in to unmask]
> > Datum: 2. 5. 2014 0:04:14
> > Předmět: PSF measurement using Au beads
> >
> > "*****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > 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"
> >
>