Confocal Microscopy List

Re: intensity measurements

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/intensity-measurements-tp3005594p3008813.html

Well my take on this is that counterstaining is just crazy if you are trying to do quantitative measurements in transmitted light wide-field.

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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Tuesday, 2 June 2009 5:43 AM
To: [hidden email]
Subject: Re: intensity measurements

The one issue I have with using a monochrome camera to image DAB stained slides is that one loses all color information. If the sample is counterstained with fort example a blue dye, it's impossible to tell dark brown features (DAB) from dark blue features. Color cameras are pretty good these days, and if you collect a color transmitted light image you still have the option later on to convert it to monochrome for intensity staining measurement, with the added benefit that you can try to extract different color channels and see which one best separates your DAB (or other) staining from your counterstain. Another benefit would be to be able to do positive cell counts: you can get a count for all cells (stained either blue or brown, for example), and then you can get a count for the positive cells (stained brown only). I'm not sure how one would do that with monochrome images.... a negative blue-counterstained cell might appear just as dark as a positive light-brown stained cell...

Julio.

On Jun 1, 2009, at 11:45 AM, Chris Tully wrote:

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
[hidden email]
240-888-1021
http://www.linkedin.com/in/christully

2009/6/1 Guy Cox <[hidden email]>

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
______________________________________________
Phone +61 2 9351 3176     Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
     http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Tuesday, 2 June 2009 2:32 AM
To: [hidden email]
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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