Re[2]: Membrane polarization

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
03 May 96 18:13:26 Z

It is also important to remember that reflection on optical
surfaces is critical to the polarization plane. Depending on
your optical arrangement it is therefore important which
detector you use for what polarization plane. To get light in
and out of a normal flow cell (flow from top to bottom or
reverse) the laser should be vertically polarized, thus your
deflectors for the vertical polarized light should deflect in
the plane of the optical bench. I always wondered but never
checked to what percentage the normal antibody fluorescence
retains the direction of polarization, thus whether there is
a disadvantage in the arrangement that Coulter has chosen to
deflect towards the ceiling with their dichroics?

Gerhard Nebe-v.Caron Unilever Research, Colworth Lab.
Sharnbrook, Beds. GB-MK44 1LQ Tel.:+44(0)1234 222066
Fax.: 222344 Gerhard.Nebe-von-Caron@unilever.com

______________________________ Reply Separator
_________________________________ Subject: Re: Membrane polarization
Author: t-delohery@ski.mskcc.org at INTERNET Date: 03/05/96 00:30

> We are trying to measure membrane polarization by flow
cytometry >using the probe TMA-DPH. So far we've had no luck. Is
anyone else doing >this? If so, can you tell me which probes you have
found most successful >and the specs on your polarization filters. We
are using a FACS Vantage >and "hand-me-down" polarizers (no specs).
The polarizers we're using are >yellow, and we're wondering if we were
just cutting out most of the >TMA-DPH emitted signal.

Besides your filters you also have to determine the plane of
polarization of your laser at the stream intercept. While the beam is
vertically polarized as it exits the laser, it may be in any direction
after passing through all the prisms on the Vantage. It may not even
be polarized.

Fluorescence polarization measurments depend on "photoselection" -
selectively exciting molecules whose absorption dipole moments are
parallel to plane of polarization of the exciting light, ie, the
laser. Molecular motion during the excited state lifetime determines
the degree of fluorescence polarization. A freely moving fluorochrome
can reorient to all possible planes during the excited state lifetime
and no fluorescence polarization is observed. An immobilized
fluorochrome at low temperature can yield highly polarized
fluorescence.

It's also extremely convenient to be able to change the lasers'
polarization for calibration purposes.

td i've been waiting years to say that.

--
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 Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center |
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