Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/intensity-measurements-tp3005594p3007426.html
In terms of quantitation, surely the best result is to
forget RGB imaging altogether. From what I recall DAB gives a browny-black
colour, so a greyscale image should do fine. But I do echo Julio's doubts
about quantitation. If you are working in fluorescence, rather than
picking one RGB channel the best thing is to get a filter (or set a spectral
range) which matches your fluorochrome and excludes anything else, then again
record an image of just that channel.
Guy
Optical Imaging Techniques in Cell Biology
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Cox CRC Press / Taylor & Francis
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I should add a couple of things:
1. several of the manufacturers who sell digital pathology systems and/or
image analysis software now have tools to separate and analyze color
images such as those obtained from IHC chromogenic stains. Some may
actually do a pretty good job at separating specific color ranges or hues. The
problem here is that often the analysis has to be limited to measuring the
stained area fraction, since in a color image, the "intensity" is a somewhat
meaningless notion (you get the average of three values, such as red, green, and
blue), and very different colors or hues may give similar "intensity" values.
That's why separating color channels first and picking the one that's most
representative is useful. In many cases though, measuring the area fraction that
has the desired color range may be just fine. It all depends on the information
that is most relevant for your experiment.
2. You also need to ask yourself whether DAB and other chromogenic
stains are quantitative. You can generally distinguish pale from medium from
dark staining, but the "dynamic range" is most probably more limited than, for
instance, fluorescent staining. Doing some staining intensity calibrations would
certainly be a good idea, to see how linear and/or quantitative this approach
is.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA
==
On Jun 1, 2009, at 3:51 AM, vaishali kailaje wrote:
hello friends,
We are interested in measuring the intensity in
immnohistochemical slides (DAB stained).
Could anybody suggest some software or a method to
quantitate DAB intensity in tissue sections.
Secondly,can these measurements be made using
metomorph, ImageJ or Axiovision as these are the softwares available with
us.
Thanks
Vaishali Kailaje
Scientific Assistant,
Tata Memorial Centre,
Advanced Centre for Treatment Reasearch Education in
Cancer
Kahrghar,
Navi Mumbai,
India.
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