Re: WB Analysis Convert Histogram area to OD

Posted by Jonathan Hilmer on Jan 17, 2009; 6:17pm
URL: http://imagej.273.s1.nabble.com/WB-Analysis-Convert-Histogram-area-to-OD-tp3694029p3694030.html

> I like the CTRL-1 , 2, 3 Gel analysis method which brings up a histogram of
> each selected lane. Then I can see the background, draw a line so that I
> know I am getting just the band of interest. However, what is that number?

I assume the number you're referring to is the automated measurement
you get with the area selection tool.  That measurement is literally
just the area of the selection, exactly as you see it.  There is some
normalization/scaling code that runs in the course of creating the
plots, so the dimensions and measurements from the lane plots do NOT
correspond directly to any kind of calibrated units.  However, they
should be linearly scaled and measurements within one plot-image
maintain appropriate relative sizes.  It's been a while since I looked
at the source code, but I think the only exception to this would be
for very small images which are not scaled down.  I doubt you'd have
plots that small, however.

It is possible to alter the code to display the scaling factors to the
user (which you would then use to correct the measurements manually
after quantitation), or remove the scaling (which generates huge plot
images) so that the area measurements reflect the actual integrated
density, but that's not part of the standard GelAnalyzer.

You can not directly quantitate -between- multiple runs of GelAnalyzer
plots with different selections, because this scaling will probably be
different for each run.  Relative quantitation needs to be all within
one ctrl-1,2,2,2,etc,3 set.

Some of the other tools, such as plot-profile and the general
measurements, are better suited to make direct measurements of the OD,
since they don't scale the numbers before presenting them to you.  The
tradeoff is that you don't have the interactive definition of
background and ROI as you do with the GelAnalyzer plots.

My personal preference has been to use modified GelAnalyzer code to
generate large plots, but without any correction to actual OD units.
The relative intensities give me the measurments I need.  However,
this is for gels with features (bands) that run very close together
and with varying background, so the definitions of band borders and
the background are very important.  If your blots have well-separated
features with low background then it would be better to just use the
measurements tool after specifying appropriate rectangular or
user-defined ROIs.

Jonathan