Kees
Software is great!!!!
I think it is also important to check your lenses to make sure the two
fluorescence colors that your are studying are indeed colocalized.  I have
observed very good and expensive lenses to be difficient in this parameter
Unless you check your system with either fluorescent beads or a single
reflecting mirror  your estimates of colocalization may be artifacts due to
improper functioning equipment. Colocalization starts with the hardware.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
MD 72
National Health and Environmental Effects Research Laboratory
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: [log in to unmask]




                    Kees Jalink <[log in to unmask]>
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                    FFALO.EDU>                        Subject:     Re: colocalization, more
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                    02/28/01 11:48 AM
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Thanks for those suggestions.

But before trying to track down all those pieces of software to run our
data through, what is the basis of quantification? More specifically,
what statistical (or other) math is used to come to a number that
describes just how well 2 images colocalize? I have tried a bit using
e.g. correlation coeeficient of paired pixel intensities, or covariance,
or Pearsons regression coeeficient, and roughly get numbers that give
what you expect. But how significant are those values (without putting a
full month into trying to calculate that)? For example, I noted that if
I make 2 images of the same channel, one noisy and the other nice and
smooth by averaging, I get correlation coefficients that are worse than
in the case that I take 2 times that smooth image, one of which is
rotated by 90 degrees (which clearly is a wrong result).

Regards, Kees

--

Kees Jalink Ph.D.
The Netherlands Cancer Institute, dept. of Cell Biology H1
Plesmanlaan 121, 1066CX Amsterdam, the Netherlands
020-5121933 (tel office)/ ...1947 (tel lab) / ...1944 (fax) /
[log in to unmask] (email)
at home: Paulus Potterlaan 5, 2102CC Heemstede
0235-476047 / [log in to unmask]