Re: PSF measurement using Au beads

Posted by Lu Yan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-measurement-using-Au-beads-tp7581962p7581980.html

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Hi Zdenek,

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
collimate several colors (from 450 nm~650 nm), but we found also that
Thorlabs achromat shifted the focal points of 532 nm and 650 nm by 600 nm
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.
objective usually does not do a lot chromatic aberration corrections so
it's kind of tricky to find one, with the right focal length.

Thanks,
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
[hidden email]
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On Fri, May 2, 2014 at 6:15 AM, Zdenek Svindrych <[hidden email]> wrote:

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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"
>