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]]
Sent: Thursday, October 02, 2008
5:38 PM
To:
[hidden email]
Subject: Re: cell membrane image
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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