Re: cell membrane image

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/fluorescent-Ab-counterstain-for-chicken-somites-lectins-tp1130451p1140317.html

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:

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 [[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