fluorescent Ab counterstain for chicken somites (lectins?)
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matthewmakes |
fluorescent Ab counterstain for chicken somites (lectins?)
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hi. i'm interested in picking up a quick, simple and fairly affordable fluorescent counterstain for viewing cell morphology in antibody labeled sections of chicken embryos. from what i can put together, some sort of lectin would likely do the job, but i'm having a hard time finding material with lectin labels used in the chicken embryo. i've seen some papers with nice wheat germ agglutinin labels in similar staged somites of mice. i primarily need this as an Ab counterstain, but if i could get something that would work live as well, that would be great. (i've got a collection of membrane labels for live imaging, but more options are always nice.) any advice would be greatly appreciated. thank you, matthew See how Windows Mobile brings your life together—at home, work, or on the go. See Now |
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Carol Heckman |
Re: fluorescent Ab counterstain for chicken somites (lectins?)
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Matthew-
I don't seen any reason why the wheat germ agglutinin labels wouldn't work. The cell membrane protein structure might be highly conserved between mouse and chicken. Carol ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Matthew Smith [[hidden email]] Sent: Tuesday, September 30, 2008 9:12 PM To: [hidden email] Subject: fluorescent Ab counterstain for chicken somites (lectins?) hi. i'm interested in picking up a quick, simple and fairly affordable fluorescent counterstain for viewing cell morphology in antibody labeled sections of chicken embryos. from what i can put together, some sort of lectin would likely do the job, but i'm having a hard time finding material with lectin labels used in the chicken embryo. i've seen some papers with nice wheat germ agglutinin labels in similar staged somites of mice. i primarily need this as an Ab counterstain, but if i could get something that would work live as well, that would be great. (i've got a collection of membrane labels for live imaging, but more options are always nice.) any advice would be greatly appreciated. thank you, matthew ________________________________ See how Windows Mobile brings your life together—at home, work, or on the go. See Now<http://clk.atdmt.com/MRT/go/msnnkwxp1020093182mrt/direct/01/> |
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Koo, Lily (NIH/NIAID) [E] |
cell membrane image
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Dear all,
When we imaging erythrocytes labeled with a membrane dye on a SP5 confocal, we noticed that in all images, the top and bottom of the cell membrane appeared much dimmer than the left and right sides of it. Is there a particular reason for this observation? Is there a difference in the resolution of the x-scan vs. y-scan? Thanks much. Lily |
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Interesting observation. Assuming the PSF is not assymetric in x-y,
could this be due to the interaction of the orientation of the dye molecules and the polarization of the laser? Regards Mark Cannell Koo, Lily (NIH/NIAID) [F] wrote: > Dear all, > > When we imaging erythrocytes labeled with a membrane dye on a SP5 > confocal, we noticed that in all images, the top and bottom of the cell > membrane appeared much dimmer than the left and right sides of it. Is > there a particular reason for this observation? Is there a difference > in the resolution of the x-scan vs. y-scan? > > Thanks much. > > Lily > |
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In reply to this post by Koo, Lily (NIH/NIAID) [E]
We see they same thing in dye labeled lipid vesicles. It has to
do with the dipole orientation with respect to the laser polarization. Taking 2 images using a quarter wave plate with one should show you equal presence. You may also see this effect if you use the beam rotator on the SP5 Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology Princeton University 609-258-5432 Visit us at http://www.molbio1.princeton.edu/facility/confocal/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Koo, Lily (NIH/NIAID) [F] Sent: Thursday, October 02, 2008 4:40 PM To: [hidden email] Subject: cell membrane image Dear all, When we imaging erythrocytes labeled with a membrane dye on a SP5 confocal, we noticed that in all images, the top and bottom of the cell membrane appeared much dimmer than the left and right sides of it. Is there a particular reason for this observation? Is there a difference in the resolution of the x-scan vs. y-scan? Thanks much. Lily |
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There have been two replies now on this potentially being a light polarization and dye photoselection/dichroism interaction. There are ways to verify this.
First, what membrane probe are you using? Is it one of the carbocyanine dyes like DiI? Dan Axelrod showed many years ago that probes like this orient a particular way in the membrane and that this effect can be exploited several ways (See, Axelrod Biophysical Journal, 1979). Try rotating your sample 90 degrees on the microscope stage and see if the cells are still lighting up the same way. If so, then you can be sure that photoselection is occurring. You can check the degree and direction of polarization of your microscope laser source by placing film polarizer (polaroid film) direction on the stage and rotating it. Remove the objective for this measurement and set the microscope to do a point scan in the middle of the scan field. If when you rotate the film with its reference direction initially pointing North-South to East-West and the laser light coming through disappears, then you can be sure that your laser beam is strongly polarized in the NS direction (and therefore photoselection would occur in the top-bottom direction of your image if the dye has a dipole that orients perpendicular to the membrane, something like DPH or Laurdan. For DiI or DiO, it would be the opposite - left and right side would preferentially light up - since the transition dipoles for this dye tend to align parallel to the membrane). Joe's suggestion of inserting a quarter-wave plate into the excitation beam and turning it 45 degrees relative to the laser beam polarization direction should get rid of this problem since by doing so you circularize the polarization of the laser beam, thus equally exciting all directions that are perpendicular to the laser beam direction of travel (ie: anywhere in the xy plane). This posting by Lily brings up the point that this effect is present all the time in all confocal microscopes - at least the ones that use laser sources (which are usually strongly polarized) and polarization-maintaining optical fibers to deliver the laser beams into the scan head. If your probes are oriented in your sample, you might unintentionally be selecting parts of your sample to light up by fluorescence. John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 On 2-Oct-08, at 5:14 PM, Goodhouse, Joseph G. wrote:
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Koo, Lily (NIH/NIAID) [E] |
Re: cell membrane image
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Sincere thanks to John and Joe’s
replies. The Polaroid film idea is neat, I will definitely try it. Thanks for
the quarter-wave plate idea. Lily From: John Oreopoulos
[mailto:[hidden email]] There have been two replies now on this potentially being a light
polarization and dye photoselection/dichroism interaction. There are ways to
verify this. First, what membrane probe are you using? Is it one of the carbocyanine
dyes like DiI? Dan Axelrod showed many years ago that probes like
this orient a particular way in the membrane and that this effect can
be exploited several ways (See, Axelrod Biophysical Journal, 1979). Try
rotating your sample 90 degrees on the microscope stage and see if the cells
are still lighting up the same way. If so, then you can be sure that
photoselection is occurring. You can check the degree and direction of polarization of your
microscope laser source by placing film polarizer (polaroid film) direction on
the stage and rotating it. Remove the objective for this measurement and set
the microscope to do a point scan in the middle of the scan field. If when you
rotate the film with its reference direction initially pointing North-South to
East-West and the laser light coming through disappears, then you can be sure
that your laser beam is strongly polarized in the NS direction (and therefore
photoselection would occur in the top-bottom direction of your image if the dye
has a dipole that orients perpendicular to the membrane, something like DPH or
Laurdan. For DiI or DiO, it would be the opposite - left and right side would
preferentially light up - since the transition dipoles for this dye tend to
align parallel to the membrane). Joe's suggestion of inserting a quarter-wave plate into the excitation
beam and turning it 45 degrees relative to the laser beam polarization
direction should get rid of this problem since by doing so you circularize the
polarization of the laser beam, thus equally exciting all directions that are
perpendicular to the laser beam direction of travel (ie: anywhere in the xy
plane). This posting by Lily brings up the point that this effect is present
all the time in all confocal microscopes - at least the ones that use laser
sources (which are usually strongly polarized) and polarization-maintaining
optical fibers to deliver the laser beams into the scan head. If your probes
are oriented in your sample, you might unintentionally be selecting parts of
your sample to light up by fluorescence. John Oreopoulos, BSc, PhD Candidate Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 On 2-Oct-08, at 5:14 PM, Goodhouse, Joseph G. wrote:
We
see they same thing in dye labeled lipid vesicles. It has to do with the dipole orientation with respect to the laser polarization. Taking 2 images using a quarter wave plate with one should show you equal presence. You may
also see this effect if you use the beam rotator on the SP5 Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology 609-258-5432 -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Sent: Thursday, October 02, 2008 4:40 PM To: [hidden email] Subject: cell membrane image Dear all, When we imaging erythrocytes labeled with a membrane dye on a SP5 confocal, we noticed that in all images, the top and bottom of the cell membrane appeared much dimmer than the left and right sides of it. Is there a particular reason for this observation? Is there a difference in the resolution of the x-scan vs. y-scan? Thanks much. Lily
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Boswell, Carl A - (cboswell) |
Re: fluorescent Ab counterstain for chicken somites (lectins?)
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In reply to this post by Carol Heckman
Hi Matthew,
As a point of clarification, lectins will bind specific sequences of sugars on glycoproteins. As such, identical proteins with different glyco-residues will have different lectin binding properties. Given that, you may need to do what I had to do years ago, and that was sample a bunch of different lectins to see what works. When lectin chemistry was new, one could find samplers with small amounts of several lectins to test. Now, I don't see any. Sigma Chemical has many lectins, though I don't know how many have been fluorescently tagged. Molecular Probes is another source of labelled lectins. Good luck, Carl Carl A. Boswell, Ph.D. Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 ----- Original Message ----- From: "Carol Heckman" <[hidden email]> To: <[hidden email]> Sent: Thursday, October 02, 2008 12:22 PM Subject: Re: fluorescent Ab counterstain for chicken somites (lectins?) Matthew- I don't seen any reason why the wheat germ agglutinin labels wouldn't work. The cell membrane protein structure might be highly conserved between mouse and chicken. Carol ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Matthew Smith [[hidden email]] Sent: Tuesday, September 30, 2008 9:12 PM To: [hidden email] Subject: fluorescent Ab counterstain for chicken somites (lectins?) hi. i'm interested in picking up a quick, simple and fairly affordable fluorescent counterstain for viewing cell morphology in antibody labeled sections of chicken embryos. from what i can put together, some sort of lectin would likely do the job, but i'm having a hard time finding material with lectin labels used in the chicken embryo. i've seen some papers with nice wheat germ agglutinin labels in similar staged somites of mice. i primarily need this as an Ab counterstain, but if i could get something that would work live as well, that would be great. (i've got a collection of membrane labels for live imaging, but more options are always nice.) any advice would be greatly appreciated. thank you, matthew ________________________________ See how Windows Mobile brings your life together—at home, work, or on the go. See Now<http://clk.atdmt.com/MRT/go/msnnkwxp1020093182mrt/direct/01/> |
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G. Esteban Fernandez |
Re: fluorescent Ab counterstain for chicken somites (lectins?)
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Vector Labs sells lectin kits.
-Esteban On Fri, Oct 3, 2008 at 1:36 PM, Carl Boswell <[hidden email]> wrote: Hi Matthew, -- G. Esteban Fernandez, Ph.D. Associate Director Molecular Cytology Core Facility University of Missouri 120 Bond Life Sciences Center Columbia, MO 65211 http://www.biotech.missouri.edu/mcc/ 573-882-4895 573-884-9395 fax |
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