http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581980.html
Thanks for your advices. We are using a multimode fiber acting as a
detection pinhole. We use an 60X/1.35 objective (olympus), f= 300 mm lens
to focus the light into detection fiber. The excitation laser line is 650
nm, so one airy unit= 1.22*650nm/1.35 * 300mm/30mm = 58.7 um. I have three
fibers with core diameters of 50 um, 100 um, 150 um. I am currently using
50 um one. I tried 150 um, the image was actually similar in terms of
overall shape, i.e. 4 lobes.
For the achromat doublet. Our intention was to use the same lens to
on the sample plane. We are thinking of changing to some other
lenses/objective lens. Do you have any suggestions? I noticed that low mag.
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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"
>