Re: PSF measurement using Au beads

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581972.html

*****
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 <[hidden email]> 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 <[hidden email]>
> Komu: [hidden email]
> 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"
>