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To make a contribution to the list of emails on forward scattering
and sizing:
For spheres in the um region (sizes similar to blood cells), forward scattering
is following mie scattering. The theory only holds for spheres.
Additions to this were made by Kerker who worked out the coated sphere
theory. This theory models the scattering of a sphere within a sphere
(the very rough model for nucleated cells).
Burger and Jett showed the use of this theory by looking at nucleated
rbc of toad and chicken (I would have to check the exact reference, might
be in Cytometry around 1988}.
Articles were published with additions to Mie theory for aspherical
particles (e.g. Sato).
We have to keep in mind that Mie theory, which describes polymer
beads in a cytometer quite well, uses the relative refractive index,
so that of beads (or for that matter,cells) compared to the flow
around the particle (bloody fluids and sheat flow combination).
At our lab we have shown that forward scattering of beads can be
modelled by diffraction, refraction and a few internal reflections
within the bead. The internal reflections cause a smear effect onto
the diffraction pattern, fast oscillations are smoothed out.
For forward scattering upto 30 dgrs., this model is quite similar to
the mie calculations we have, showing that simple optics can help us
save a lot of calculation time. We are still working on it so it is
not available yet on the net.
The diffraction part would give the best size information.
If anyone needs to do sizing experiments, I would suggest keeping the
collected forward scatterangle small, just around the obscuration
bar,eg. 1-3 dgrs. In that way a large part of the diffraction lobe
can be measured but the smoothing effect will not effect the
measurement.
In many commercial cytometers this means you should
should get some black tape and put it over the sides of the forward
scatter detector (or if the obscuration bar is not directly on the
detector, put the tape just beside the obscuration bar holder).
Keep a small area on both sides of the obscuration bar open.
If anyone has a cytometer with a round obscuration device, make a
ring or first make a bar from it).
If anyone is interested in the exact references of the articles
mentioned earlier, let me know, I will look them up.
Have fun taping.
Robbert van Leeuwen
Applied Optics / Flowcytometry Group
Dept. of Applied Physics
University of Twente
The Netherlands.
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