PSF measurement using Au beads
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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 |
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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, It seems to me that reflecting beads would be a tricky object to get PSF from because you have to deal with the angular dependence of scattering and reflection. 8 um might be the distance between a slide and a coverslip, both surfaces should reflect. Mike Model ________________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Lu <[hidden email]> Sent: Thursday, May 01, 2014 5:49 PM To: [hidden email] Subject: 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 |
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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 Mike, Thanks for your reply. Yes you are right those two layers are cover glass and glass slide. I have seen in many papers people using gold beads to probe the focal intensity distribution which somewhat related to the PSF of the system, so I just figured it might be easier to measure in this way, and i wanted to know if they had similar problems. But this distorted PSF seems to be related to the fact that the incident beam has non perpect Gaussian profile, which confuses me most. Thanks, Lu On May 1, 2014 6:35 PM, "MODEL, MICHAEL" <[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, > > It seems to me that reflecting beads would be a tricky object to get PSF > from because you have to deal with the angular dependence of scattering and > reflection. 8 um might be the distance between a slide and a coverslip, > both surfaces should reflect. > > Mike Model > > ________________________________________ > From: Confocal Microscopy List <[hidden email]> on > behalf of Lu <[hidden email]> > Sent: Thursday, May 01, 2014 5:49 PM > To: [hidden email] > Subject: 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 > |
 
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Kyle Michael Douglass |
Re: PSF measurement using Au beads
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yan, Lu
Sent: vendredi 2 mai 2014 03:24 To: [hidden email] Subject: Re: PSF measurement using Au beads On May 1, 2014 6:35 PM, "MODEL, MICHAEL" <[hidden email]> wrote: From: Confocal Microscopy List <[hidden email]> on behalf of Lu <[hidden email]> Sent: Thursday, May 01, 2014 5:49 PM To: [hidden email] Subject: PSF measurement using Au beads 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? It seems to me that reflecting beads would be a tricky object to get PSF from because you have to deal with the angular dependence of scattering and reflection. 8 um might be the distance between a slide and a coverslip, both surfaces should reflect. Thanks for your reply. Yes you are right those two layers are cover glass and glass slide. I have seen in many papers people using gold beads to probe the focal intensity distribution which somewhat related to the PSF of the system, so I just figured it might be easier to measure in this way, and i wanted to know if they had similar problems. But this distorted PSF seems to be related to the fact that the incident beam has non perpect Gaussian profile, which confuses me most. Hi Lu, I think that Mike is right about having to consider the angular dependence of scattering from the beads. (I'm assuming that your beam is collimated at the back focal plane). In confocal setups you illuminate your bead with a number of plane waves (i.e. k-vectors) that span a solid cone. Each plane wave within this cone is going to independently scatter light into a pattern that you can determine using Mie theory. Because the scattering is coherent, the total scattered field is just the sum of the different field scattering profiles from all the plane waves traveling in different directions within the cone, and the intensity that you measure is a projection of the squared-field distribution that fits within your system's numerical aperture onto a plane. All that being said, if you reduce the size of your beam in the back focal plane of the objective, then this is equivalent to illuminating your beads with a cone of light with a smaller apex angle. Equivalently, you have fewer plane waves that scatter and the total scattered field is summed over a smaller number of Mie field profiles. I suspect that this reduces any interference effects in the total scattered field and is what eliminates the lobes you're observing. To summarize, the messy axial profile might not originate from a dirty excitation beam, but simply because you're exciting the sphere with a number of plane waves traveling in different directions and the scattered fields from each of these plane waves is are interfering. If the spheres are touching the glass, this could also introduce asymmetries in the profile but I doubt it since you mentioned the spheres are mounted in glycerol, which has a refractive index close to glass. You could check the Mie profiles with this handy webapp: http://omlc.ogi.edu/cgi-bin/mie_angles.cgi?diameter=0.15&lambda_vac=0.650&n_medium=1.47&nr_sphere=0.18&ni_sphere=-3.42&n_angles=100&density=0.1 I already entered the material parameters based on what you mentioned, but you should double check them anyway. Good luck! Kyle Dr. Kyle M. Douglass Postdoctoral Fellow The Laboratory of Experimental Biophysics EPFL, Lausanne, Switzerland +41 21 69 30556 (Office) |
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Doube, Michael |
Re: PSF measurement using Au beads
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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, We've done something like this in the past, but with 80nm (IIRC) Au particles embedded in agarose and imaged with a water immersion objective (upright Zeiss LSM 780 NLO, testing alignment of all laser lines). I don't recall that the PSF was as exotic as yours - it looked like an ordinary 'Airy disk' fluorescence PSF. Michael <http://www.rvc.ac.uk> This message, together with any attachments, is intended for the stated addressee(s) only and may contain privileged or confidential information. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Royal Veterinary College. |
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Zdenek Svindrych |
Re: PSF measurement using Au beads
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In reply to this post by Lu Yan
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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" |
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Craig Brideau |
Re: PSF measurement using Au beads
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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" > |
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In reply to this post by Kyle Michael Douglass
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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 Kyle, Thanks for your post. We have thought about the Mie scattering. It will indeed affecting the measurement, but we did not expect to see that pronounced 'distortion'. In Ch.11 of Pawley's Handbook of Confocal Micro., Juskaitis describes several methods to measure 'true' PSF of objective lenses including phase information. There I think they were also using gold beads as sample. So I was wondering if my case is due to some other things, like I mentioned in the post that maybe the collimation lens does a so bad job to create a Gaussian beam with nearly flat phase at around the back aperture of my objective, such that a lot of other spatial modes also get to enter into the objective to cause unexpected 'PSF'. The webapp is really helpful. Thanks for that again. Cheers, 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] ----------------------------------------------------- On Fri, May 2, 2014 at 3:28 AM, Douglass Kyle Michael <[hidden email] > wrote: > From: Confocal Microscopy List [mailto: > [hidden email]] On Behalf Of Yan, Lu > Sent: vendredi 2 mai 2014 03:24 > To: [hidden email] > Subject: Re: PSF measurement using Au beads > > On May 1, 2014 6:35 PM, "MODEL, MICHAEL" <[hidden email]> > wrote: > > From: Confocal Microscopy List < > [hidden email]> on > behalf of Lu <[hidden email]> > Sent: Thursday, May 01, 2014 5:49 PM > To: [hidden email] > Subject: PSF measurement using Au beads > > 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? > > It seems to me that reflecting beads would be a tricky > object to get > PSF from because you have to deal with the angular > dependence of > scattering and reflection. 8 um might be the distance > between a slide > and a coverslip, both surfaces should reflect. > > Thanks for your reply. Yes you are right those two layers are > cover glass and glass slide. I have seen in many papers people using gold > beads to probe the focal intensity distribution which somewhat > related to the PSF of the system, so I just figured it might be easier to > measure in this way, and i wanted to know if they had similar > problems. But this distorted PSF seems to be related to the fact that the > incident beam has non perpect Gaussian profile, which confuses me most. > > Hi Lu, > > I think that Mike is right about having to consider the angular dependence > of scattering from the beads. (I'm assuming that your beam is collimated at > the back focal plane). In confocal setups you illuminate your bead with a > number of plane waves (i.e. k-vectors) that span a solid cone. Each plane > wave within this cone is going to independently scatter light into a > pattern that you can determine using Mie theory. Because the scattering is > coherent, the total scattered field is just the sum of the different field > scattering profiles from all the plane waves traveling in different > directions within the cone, and the intensity that you measure is a > projection of the squared-field distribution that fits within your system's > numerical aperture onto a plane. > > All that being said, if you reduce the size of your beam in the back focal > plane of the objective, then this is equivalent to illuminating your beads > with a cone of light with a smaller apex angle. Equivalently, you have > fewer plane waves that scatter and the total scattered field is summed over > a smaller number of Mie field profiles. I suspect that this reduces any > interference effects in the total scattered field and is what eliminates > the lobes you're observing. > > To summarize, the messy axial profile might not originate from a dirty > excitation beam, but simply because you're exciting the sphere with a > number of plane waves traveling in different directions and the scattered > fields from each of these plane waves is are interfering. If the spheres > are touching the glass, this could also introduce asymmetries in the > profile but I doubt it since you mentioned the spheres are mounted in > glycerol, which has a refractive index close to glass. > > You could check the Mie profiles with this handy webapp: > http://omlc.ogi.edu/cgi-bin/mie_angles.cgi?diameter=0.15&lambda_vac=0.650&n_medium=1.47&nr_sphere=0.18&ni_sphere=-3.42&n_angles=100&density=0.1 > > I already entered the material parameters based on what you mentioned, but > you should double check them anyway. > > Good luck! > > Kyle > > Dr. Kyle M. Douglass > Postdoctoral Fellow > The Laboratory of Experimental Biophysics > EPFL, Lausanne, Switzerland > +41 21 69 30556 (Office) > > > |
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In reply to this post by Doube, Michael
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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 Michael, We use 150 nm (Cytodiagnostics). We saw some nice Airy disk PSF long before with another setup. We just diluted it at 1:1000 with ethanol, shake it well, and then 1:1000 again with ethanol. Actually I got another question for sample preparation, is there a sort of standard gold beads sample preparation procedure that one can follow? My beads tend to form smaller clusters/islands all over the cover glass (not necessarily dense) after the ethanol evaporated off. Do I need to spin coat them? 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] ----------------------------------------------------- On Fri, May 2, 2014 at 3:40 AM, Doube, Michael <[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, > > We've done something like this in the past, but with 80nm (IIRC) Au > particles embedded in agarose and imaged with a water immersion objective > (upright Zeiss LSM 780 NLO, testing alignment of all laser lines). I don't > recall that the PSF was as exotic as yours - it looked like an ordinary > 'Airy disk' fluorescence PSF. > > Michael > > <http://www.rvc.ac.uk> > > This message, together with any attachments, is intended for the stated > addressee(s) only and may contain privileged or confidential information. > Any views or opinions presented are solely those of the author and do not > necessarily represent those of the Royal Veterinary College. > |
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In reply to this post by Zdenek Svindrych
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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 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] ----------------------------------------------------- On Fri, May 2, 2014 at 6:15 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" > |
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In reply to this post by Craig Brideau
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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. ***** But would this scattered photon really mess up the psf measurement? ----------------------------------------------------- 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] ----------------------------------------------------- On Fri, May 2, 2014 at 4:42 PM, Craig Brideau <[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. > ***** > > 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" > > > |
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James Pawley |
Re: PSF measurement using Au beads
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In reply to this post by Lu Yan
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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, As a great fan of what I like to call "backscattered light imaging" (BSL), and also of using gold beads to determine the PSF, I am intrigued by your problems. You mention that the specimen has only glass or oil between the objective and the focus plane. That is good. However, if one of your paired images relates to light light reflecting at the glycerol/glass surface on the far side of the particle, it would have considerable SA because of the light having passed twice through some microns of glycerol. Remember that in the Handbook chapter that you mentioned, Juskaitis found that he got SA even using oil lenses used with the correct oil if the oil was the wrong temperature or the coverslip was the wrong thickness. It is all very sensitive. In fact, I admit to being surprised that you can use BSL to see a bead at a glass/glycerol interface at all. You should have an immense signal from the glass/glycerol interface that would swamp anything you get from a submicron gold bead. (This reflected light signal will have more intensity at high-NA angles because the reflectivity at an RI interface increases strongly as the angle between the ray and the surface becomes smaller. This may be related to your observation that there is less problem when you close down your objective.) Embedding the beads in the proper immersion oil should reduce this problem. In terms of the "double image," I think we need to look at the effects of reflections, particularly those from the flat or nearly flat optic al surfaces such as the ends of the fibres and the slide/coverslip. I didn't see any mention of efforts to use the normal "antiflex" techniques that use polarizers and 1/4 wave plates to discriminate against signals caused by reflections from the optical components such as the scan lens and the objective. I clearly remember the problems we had trying to get rid of reflections that seemed to originate from in the Nikon widefield eyepiece used for a scan lens in the BioRad MRC confocals. Reflections from curved surfaces have virtual origins and if the optics between this origin and the pinhole (sensor-fibre) plane have the effect of focusing this virtual origin onto the sensor plane, will create a "blobby" artifact. Of course, our blobs didn't go in and out of focus as you move the objective in z so it is unlikely that this is the exact cause of the artifact that you are seeing, but it is the only example that I have. As you seem to have a copy of The Handbook available, you might look at the pages shown in the Index under the headings "backscattered light" and "antiflex". And you might try looking at some larger beads... Good luck, Jim Pawley >***** >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 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] >----------------------------------------------------- > > >On Fri, May 2, 2014 at 6:15 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 >> >> >> >> ---------- PÛvodní zpráva ---------- >> Od: Lu <[hidden email]> >> Komu: [hidden email] >> Datum: 2. 5. 2014 0:04:14 >> PÞedmût: 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" >> -- **************************************** James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146 |
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Zdenek Svindrych |
Re: PSF measurement using Au beads
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In reply to this post by Lu Yan
*****
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, I was first puzzled by your Airy disk formula, the I spotted the typo, the last number (the objective focal length) should read 3mm, not 30mm. The result is correct, anyway. Since the PSF is still distorted, I would suspect the excitation path... For the achromat doublet. The chromatic focal shift of the lens itself ( http://www.thorlabs.com/images/popupimages/AC254-030-A_FLShift.xlsx) is about 120um between the two wavelengths you've mentioned. The axial magnification is m^2 = (30mm/3mm)^2 = 100, up to a factor of n (the medium refractive index; I never know whether to multiply or divide...). That gives a focal shift of 1.2um in sample space, it explains the shift you observe... Consider switching to 100mm achromat: the focal shift is nearly the same for a Thorlabs doublet (I would expect better figures, but the chromatic shift is optimised for shorter wavelengths). But since the magnification is now larger, you get 10 times smaller chromatic shift in the sample space. You fill the back aperture more evenly at the cost of intensity loss. Also the weaker lens is closer to a thin lens. I also remember having problems with curved dichroics. The reflected beam may get heavily distorted if the dichoic is warped. Consider TIRF-grade filters in case you need diffraction-limited performance for both excitation and emission paths. Good luck! zdenek ---------- Původní zpráva ---------- Od: Yan, Lu <[hidden email]> Komu: [hidden email] Datum: 5. 5. 2014 23:04:31 Předmět: Re: 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. ***** 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] ----------------------------------------------------- On Fri, May 2, 2014 at 6:15 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 > 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 > 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 > 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" >" |
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