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>We are having a similar problem with eGFP/eYFP unmixing, so we would be
>interested to hear any suggestions about it too.
>
>Thanks
>
>Hugo Caldas
>Columbus Children's Hospital
>Columbus, OH


Contrary to much earnest belief. META is not magic.

The ability to "unmix" depends on, among other things, having a
relatively noise free signal.  However, when the total signal in each
pixel is commonly <<100 counts, and therefore the signal from each
wavelength in one pixel is much smaller. Assuming 10 counts in a
single channel, Poisson noise will produce 30% uncertainty in this
measurement. This level of noise can make accurate fitting impossible.

Of course, when one channel has very little signal, the Poisson noise
gets worse and if you try to make a ratio from such data, it will
look awful.  It is awful.

A better plan is to smooth the data spatially (preferably in 3D) in
each wavelength channel, first. Then do the unmixing in each pixel on
the smoothed data. Assuming that your smoothing algorithm effectively
averages over 25 voxels, the S/N ratio will be 5x better and you
should get less noise.

Cheers,

Jim Pawley
--
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Prof. James B. Pawley,                             Ph.  608-263-3147
Room 223, Zoology Research Building,               FAX  608-265-5315
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"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39