Stanley's ICA, Intensity Correlation Analysis; ICQ, Intensity Correlation Quotient

[Daniel White]

Dear Elise,
Nice to hear from you on the imageJ list!

We recently reimplemented your ICA method in the new imageJ colocalisation
analysis tool, called coloc_2.
You can find it as part of the Fiji distribution of ImageJ, in the analyze
menu. Fiji in some ways superceeds the no longer supported plugin
collections from MBF / WCIF, including the old colocalisation analysis
plugins which are now obsolete and in places unfortunately buggy. Coloc_2
is automatically unit tested for proper output, so we know if its gets
broken by and updates, and we have validated the maths and caught nasty
edge cases the old plugins missed.

see
http://fiji.sc/Coloc_2

We knew no better than to cal the method "Li's ICA"
which turns out to be wrong, if your name should be used to label it.
We can fix that, if you like... and maybe, since the page
http://fiji.sc/Coloc_2
is in a wiki, you could even add your own thoughts and docs for the ICA
method there, since you know it best!
That would be a nice birthday present for the method....?
Maybe also a sensible unit test for the maths too...?

Perhaps we need to emphasise the difference between colocalisation and
covariance?

cheers

Dan



Date:    Tue, 11 Mar 2014 13:01:38 -0700
From:    EliseStanley <[hidden email]>
Subject: Stanley's ICA, Intensity Correlation Analysis; ICQ, Intensity
Correlation Quotient

Hi all,

This is my first post on the ImageJ forum.  I joined because I just
discovered that there have been a number of queries on double-fluorescence
staining analysis using the Intensity Correlation Analysis (ICA) method.
This method seems to have proved useful in many studies and has been cited
in over 300 papers since its inception (including many high-profile
reports).  Curiously, in all this time and despite its wide-ranging use its
been very rare that I have been contacted to discuss or present the method
itself.  Thus, on its 10th anniversary I thought it might be timely to come
out of hiding!

ICA was first reported, discussed and evaluated in a paper in Journal of
Neuroscience back in 2004:

[Li, Q.*, Lau, A.* Morris, T.J.*, Guo, L., Fordyce, C. and Stanley, E.F.#  A
syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve
terminal: Analysis by quantitative immunocolocalization. J. Neurosci., 24
(2004) 4070-4081.  *equal contribution; #corresponding author.

The authors were technicians (Q Li; L Guo) or trainees in my (A. Lau, T.J.
Morris) or a colleagues lab (C.Fordyce).  These co-authors did an
outstanding job generating the immunostaining and other data that form the
basis of the paper that I applied the ICA method to.  However, as my
coauthors would confirm, the method itself, its rationalization and test and
its discussion were entirely my work.

As conceived ICA was effective - but laborious since all the computations
had to be done serially on a calculator.  Because of this I asked Tony
Morris, then the manger of the Wright Cellular Imaging facility at our
facility (Toronto Western Research Institute) and an ImageJ expert (I am
sure many of you know of his contribiutions) if he could automate the
analysis.  The result was the ImageJ plugin which was first introduced in a
paper that we coauthored:
[Khanna, R., *Li, Q., Li Sun, Collins, T.J. and Stanley, E.F.#  N type Ca
channels and RIM scaffold protein covary at the presynaptic transmitter
release face but are components of independent protein complexes.
Neurosci., 140 (2006) 1201-8. *Equal contribution.]

Thus, while I conceived of the method I think we are all indebted to Tony
for making it generally available.  He also cleverly and independently
included other costaining indices in the results outcome for the convenience
of the user.

A couple of notes:
First, ICA/ICQ analysis does NOT test for colocalization (despite the
reference in the title of the paper).  The best way to illustrate this is to
use a test area that is maximally stained by both fluorophores.  The
resulting ICQ value is 0.  What
ICA/ICQ test for is covariance - if when one stain goes up the other varies
in synchrony.  In a sense, ICA tests for protein complexes in situ (see the
Khanna et all paper referred to above).

Second, I do not consider it valid to use Pearsons for analysis of
immunostaining covariance. For a discussion please see the original paper.
The reason that for immunostaining the opposite of covariance is NOT inverse
variance (as it is for Pearson's) but RANDOM staining (a negative
correlation is almost unheard of in biology - one example might be two types
of subunits that contribute to a four stave ion channel).  Indeed, this was
one of the main motivations for the invention of ICA.

Third, a tool is only as good as its wielder.  Please be careful about the
selection of areas to apply ICA analysis to.  The area should be one within
which it is POSSIBLE for the proteins to be random - such as cytoplasm of a
cell.  tireless work not only this plug-in but all of the amazing ImageJ
platform would not be possible.

I am of course very pleased with the general acceptance of this method.  I
supported its release on ImageJ (rather than selling rights) to make sure it
would be generally available to the scientific community.  It would be nice
to see the method also on commercial platforms - but that is of course
outside my control.

Finally, I wish to give my Big Thanks to Wayne Rasband - my ex-colleague at
NINDS - without who's
tireless dedication the ImageJ platform would obviously not exist.

Elise F. Stanley PhD
Senior Scientist
Toronto Western Research Institute

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