Spinning disk 20x
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I understand that the pinholes on a spinning disk instrument are optimized for high NA objectives. What does that mean optically or in terms of performance when using a low mag/low NA objective? Thanks- Dave
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Hi Dave, There’s a pretty simple calculation
to apply here to tell you if the objective “matches” the spinning
disk. The pinholes in the yokogawa spinning disk are 50um. For a 20x/0.7NA
lens, the Airy disk will be about 0.88um in diameter (the resolution as
determined by r = 0.61(wavelength)/NAobj, for GFP emission is half the diameter
of the Airy disk). The 20x will magnify this to 17.6um at the pinhole, meaning
that multiple airy disks will fit in one pinhole (equivalent to having the
pinhole open on a raster scanning confocal for example). Therefore the optical
sectioning and resolution will suffer. There is a second problem that your
camera will almost certainly undersample this in x,y, but that’s a different
discussion. For 100x/1.4NA, the Airy disk is 44um at the pinholes (for GFP),
which is why resolution is good in these conditions. Having said that, you should try different
objectives and empirically find the one which provides the information you are
after as the optical sectioning might not be so important. I hope this is helpful. I can send you a
pdf of a methods paper we wrote describing this in more detail if you like. Paul Paul S. Maddox, PhD From: I understand that the pinholes on a spinning disk instrument are
optimized for high NA objectives. What does that mean optically or in
terms of performance when using a low mag/low NA objective? Thanks- Dave Dr. David Knecht
Department of Molecular
and Cell Biology Co-head Flow Cytometry
and Confocal Microscopy Facility U-3125 860-486-2200 860-486-4331 (fax)
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Dear All,
We just had a presentation from Olympus about their LV200 bio-luminescence microscope. As they claim (and demonstrated with some images) this system is significantly (approx 10x) times more sensitive then a conventional microscope used with the same objective and camera (and pixel size/resolution). Olympus argues that the "secret" is that they put the tube lens close to the objective (probably less important) and put the camera very close to the tube lens meaning that they use a high-numerical aperture tube lens. Now I simply don't understand why this should result in a significantly higher detection intensity (and the Olympus representative was also unable to give a detailed explanation). Does anyone of you have an idea why a high NA tube lens would be advantageous? And if this is so nice - why it is not applied in conventional microscopes? Thanks Gabor |
 
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kspencer007 |
Fluorescence anisotropy on a microscope
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In reply to this post by Paul Maddox
Hello all; We
are trying to do some fluorescence polarization assays on an upright scope.
These experiments have been published using fluorescence plate readers with a
conventional dichroic cube, but we’d like to try them on a scope with a
conventional CCD. The publication does not give details on instrumentation,
other than a diagram showing a typical reflected epi-fluorescence pathway. Basically,
we are polarizing the excitation light from a xenon lamp with a fixed
polarizer. The emission light is either crossed or parallel polarized with an
adjustable analyzer right before the camera. The idea is that light from fixed
fluorescently-tagged proteins will be blocked by the crossed polarizers,
but freely moving fluorescent proteins will be detected, as their polarization dipole
is moving. Nice
idea, but in practice, it’s not really working. The polarizers really are
crossed (as determined by holding them up to room light). The crossed signal is
dimmer, but never really dark on fixed slides or Chroma’s fluorescent
test slides (no motion). The Wallaston prism is not in the light path. The
objectives are strain-free PlanApos. I’m assuming that the dichroic in the
fluorescence cube is altering the polarization? Is
there a reason why I cannot find this type of study done on a conventional
fluorescence microscope? I see that this works with MPLSM, but why not
wide-field? Our CLSM FV500 does not have proper access for me to put in the rotating
polarizers where I need them, therefore I’m trying wide-field. Please
help me to understand why this isn’t working like it does on paper. Thanks
in advance. Kathy |
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Hi Kathy,
I don't think you should expect complete anisotropy even when the fluorophore is immobilized. Emission should be more polarized than when the protein is free to tumble but never perfectly polarized - and that's what
you are observing. Besides, if you are using a high-power objective, there is some additional depolarization at the focus.
Mike Model
From: Confocal Microscopy List [[hidden email]] On Behalf Of Kathryn Spencer [[hidden email]] Sent: Thursday, May 28, 2009 4:57 PM To: [hidden email] Subject: Fluorescence anisotropy on a microscope Hello all; We are trying to do some fluorescence polarization assays on an upright scope. These experiments have been published using fluorescence plate readers with a conventional dichroic cube, but we’d like to try them on a scope with a conventional CCD. The publication does not give details on instrumentation, other than a diagram showing a typical reflected epi-fluorescence pathway. Basically, we are polarizing the excitation light from a xenon lamp with a fixed polarizer. The emission light is either crossed or parallel polarized with an adjustable analyzer right before the camera. The idea is that light from fixed fluorescently-tagged proteins will be blocked by the crossed polarizers, but freely moving fluorescent proteins will be detected, as their polarization dipole is moving. Nice idea, but in practice, it’s not really working. The polarizers really are crossed (as determined by holding them up to room light). The crossed signal is dimmer, but never really dark on fixed slides or Chroma’s fluorescent test slides (no motion). The Wallaston prism is not in the light path. The objectives are strain-free PlanApos. I’m assuming that the dichroic in the fluorescence cube is altering the polarization? Is there a reason why I cannot find this type of study done on a conventional fluorescence microscope? I see that this works with MPLSM, but why not wide-field? Our CLSM FV500 does not have proper access for me to put in the rotating polarizers where I need them, therefore I’m trying wide-field. Please help me to understand why this isn’t working like it does on paper.
Thanks in advance. Kathy
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John Oreopoulos |
Re: Fluorescence anisotropy on a microscope
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Widefield fluorescence polarization microscopy has been used off and on for quite a while in the literature. The most often cited source on this topic is Dan Axelrod's Biophysical Journal article from 1979 (also see his Methods in Cell Biology book chapter from 1989). M. Model is right about the depolarization due high NA objecive lenses. Axelrod's puplications discuss ways to account for this effect, but they assume some form of geometric model of the sample under study. I too would also expect that frozen/fixed fluorscent molecules would exhibit a small but finite depolarization since a perfect anisotropy value of 0.4 results only when the absorption and emission transition dipole moments are perfectly colinear, which is seldom the case. The presence of homoFRET can also lead to depolarization. You mentioned the possibility that your dichroic mirror might be affecting the measurement; yes, in general, anytime the light passes through any optical element, the polarization could change. This is why the analyzer is placed as the last optic right before the camera. In addition to all of the sources of depolarization mentioned above, you must also measure and correct for the instrument's intrinsic polarization bias which is corrected by finding the "G" factor, just as is done for a fluorescence spectrometer measuring polarization anisotropy (see Lacowicz's book on fluorescence spectroscopy). Contact me off the listserver if you'd like a simple protocol. John Oreopoulos
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Satyajit Mayor |
Re: Fluorescence anisotropy on a microscope
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In reply to this post by kspencer007
Dear Kathryn:
We have implemented this assay on a conventional fluorescence microscope many years ago (Varma and Mayor, Nature 1998) and still use it routinely (Sharma et al, Cell 2004). The trick is to have a very stable light source as you shift your emission polarization, in addition you must use low NA optics (< 1 NA) to get a maximum excitation/emission polarization. Do contact me off line if you want the gory details. Best Jitu Kathryn Spencer wrote: > > Hello all; > > We are trying to do some fluorescence polarization assays on an > upright scope. These experiments have been published using > fluorescence plate readers with a conventional dichroic cube, but we’d > like to try them on a scope with a conventional CCD. The publication > does not give details on instrumentation, other than a diagram showing > a typical reflected epi-fluorescence pathway. > > Basically, we are polarizing the excitation light from a xenon lamp > with a fixed polarizer. The emission light is either crossed or > parallel polarized with an adjustable analyzer right before the > camera. The idea is that light from fixed fluorescently-tagged > proteins will be blocked by the crossed polarizers, but freely moving > fluorescent proteins will be detected, as their polarization dipole is > moving. > > Nice idea, but in practice, it’s not really working. The polarizers > really are crossed (as determined by holding them up to room light). > The crossed signal is dimmer, but never really dark on fixed slides or > Chroma’s fluorescent test slides (no motion). The Wallaston prism is > not in the light path. The objectives are strain-free PlanApos. I’m > assuming that the dichroic in the fluorescence cube is altering the > polarization? > > Is there a reason why I cannot find this type of study done on a > conventional fluorescence microscope? I see that this works with > MPLSM, but why not wide-field? Our CLSM FV500 does not have proper > access for me to put in the rotating polarizers where I need them, > therefore I’m trying wide-field. > > Please help me to understand why this isn’t working like it does on paper. > > Thanks in advance. > > Kathy > -- Satyajit Mayor Professor Cellular Organization and Signalling Group National Centre for Biological Science (NCBS) Bellary Road Bangalore 560 065 Karnataka India Ph: ++91 80 2366 6260 Fax:++91 80 2363 6662 e-mail: [hidden email] web: http://www.ncbs.res.in/~faculty/mayor.html |
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Alessandro Esposito |
Re: Fluorescence anisotropy on a microscope
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In reply to this post by kspencer007
Dear Kathryn,
there is no reason not to use a wide-field system for polarization anisotropy imaging. I am trying to address some of your concerns: - reflected light could keep its polarization at a large extent, but fluorescence cannot. Therefore, with the use of one photon excitation, the maximum anisotropy one can measure is 0.4 not 1, i.e. you will always detect signal - the usefulness of polarization anisotropy is expressed when you make a ratiometric measurement; if you just measure the light passing through an orthogonal analyzer one can never know if the fluorophore is at higher concentrations or if more depolarized. If this was done on a plate reader, I could infer they were using an homogeneous assay where fluorophore concentration is fixed - multi-photon systems provide an advantage in anisotropy imaging because the fluorescence emitted by your sample can exhibit much higher anisotropies in absence of rotation and energy transfer (0.57 vs 0.4). Furthermore, a multi- photon system provide pulsed excitation and permits to perform time-resolved anisotropy. TR-FAIM allows for more information to be acquired and higher dynamic range - I do not have references at hand for wide-field systems, although you may find information looking for papers by Axelrod, Jovin, Gerritsen groups or checking Lakowicz's book; however, the wide-field system I remember was published by Rizzo and Piston on Biophys J (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1305173). If you need more information, I could send you an unpublished work where I describe a setup for anisotropy. For a wide-field system you may use a dual-view which splits the two orthogonally polarized images on the CCD camera and allow one to perform polarization anisotropy with a single detector I hope this is enough for now, Best regards, Alessandro Esposito Laser Analytics Group, Cambridge University www.quantitative-microscopy.org www.wikiscope.org |
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