Posted by
Zdenek Svindrych on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581968.html
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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
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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"