Re: intensity measurements

Posted by RICHARD BURRY on
URL: http://confocal-microscopy-list.275.s1.nabble.com/intensity-measurements-tp3005594p3008318.html

A very good solution to this issue was published last year.

Journal of Histochemistry and Cytochemistry  56 (4): 313-328, 2008

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging  by Chris M. van der Loos

This does use a spectral camera from CRI, but the method works.
 
Dick

----- Original Message -----
From: Chris Tully <[hidden email]>
Date: Monday, June 1, 2009 2:46 pm
Subject: Re: intensity measurements
To: [hidden email]

> A quick Google search on "DAB staining in optical density" turned up the following link as the first hit:

> http://www.jhc.org/cgi/reprint/42/8/1143.pdf

> A 1994 article comparing Nickle enhanced DAB staining with Flow Cytometry.  The main thing to keep in mind when analyzing images of DAB, in my opinion, is that DAB (whether Nickle enhanced or not) is a light ABSORBER, and so MUST be imaged in transmission.  When imaged in transmission, you should then use an Optical Density (OD) calibration for your pixel values.  OD varies between 0 (white) to 3 (black).  The best way to calibrate an image is to include true black (e.g. aluminum foil) and true white (no sample, cells, dust, etc.) in the sample image.  When this is not possible, the next best solution is to image these conditions seperate from the sample, but during the same session on the same scope. 

> As mentioned by Guy Cox, I would strongly recommend against using a color camera for DAB analysis.  The color filters built into any color camera will muck with the OD response of the CCD!  Use a monochrome camera and pick the exposure time (or integration time) to get a histogram that is not saturated at eaither end.

> I hope these comments are useful!

> Chris Tully


> Chris Tully
> Microscopy and Image Analysis Expert
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> 240-888-1021
> http://www.linkedin.com/in/christully

> 2009/6/1 Guy Cox <<A href="javascript:main.compose('new','t=[e-mail]')">guy@...>

> 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
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
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> From: Confocal Microscopy List [mailto:<A href="javascript:main.compose('new','t=[e-mail]')" target=1>CONFOCALMICROSCOPY@...] On Behalf Of Julio Vazquez
> Sent: Tuesday, 2 June 2009 2:32 AM
> To: <A href="javascript:main.compose('new','t=[e-mail]')" target=1>CONFOCALMICROSCOPY@...
> Subject: Re: intensity measurements

> 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 

> http://www.fhcrc.org

> ==



> 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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