Threshold methods, how to choose suitable one?

[Sarah Mohamed]

Hi

I would like to ask about the suitable choice for threshold method to quantify the area fraction of brown color.

I have H/DAB images for treated groups. I need to use a method that is the least subjective, gives consistent results meaning if i use it several times gives the exact same area fraction.

There are several methods:

1, Image>>adjust>>threshold, moving brightness and hue sliders to get 2 areas then divide them to get area fraction.

2. Image>>color>>split. choose green chanel. Image>>adjust>>threshold>>clt+M

3. www.med.upenn.edu/cellbio/documents/imagej_colorsegmentpdf.pdf

Image.. Plugins..threshold color...move saturation slider to select ROI..close plugin..image..type...8bits..image..adjust...t hreshold..clt+M.

I know there may be other methods for color segmentation but thats all i know. I cant use color deconvolution as i cant do background correction calculation ...i am just a pharmacologist.

Thanks in advance.
Sarah M.Mosaad, M.Sc.

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[Cameron Nowell-2]

Hi Sarah,

If you have the Fiji distribution of imageJ it has an option for colour
deconvolution. Go to image --> colour --> colour deconvolution. Chose H DAB
as the vectors and you will end up with 3 images. Colour 1 will be the
haemotoxylin channel, Colour 2 will be the DAB channel and Colour 3 is kind
of a remainder.

You can then threshold the DAB channel as a normal single channel image and
get out area measurments.

BIG CAVEAT - DO NOT use any of the intensity measurements as a function of
DAB levels. DAB (and other chromogens) are not linear in any way and do not
reflect expression levels in a way that can be quantified. Measuring area
and counting positive objects is fine but anything else if not possible.


Cheers

Cam



Cameron J. Nowell
Research Facilities Manager

Imaging, FACS and Analysis Core
Monash Institute of Pharmaceutical Sciences
Monash University
399 Royal Parade
(Mail address: 381 Royal Parade)
Parkville, VIC, 3052
Australia

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-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Sarah
Mohamed
Sent: Friday, 16 January 2015 5:37 AM
To: [hidden email]
Subject: Threshold methods, how to choose suitable one?

Hi

I would like to ask about the suitable choice for threshold method to
quantify the area fraction of brown color.

I have H/DAB images for treated groups. I need to use a method that is the
least subjective, gives consistent results meaning if i use it several times
gives the exact same area fraction.

There are several methods:

1, Image>>adjust>>threshold, moving brightness and hue sliders to get 2
areas then divide them to get area fraction.

2. Image>>color>>split. choose green chanel. Image>>adjust>>threshold>>clt+M

3. www.med.upenn.edu/cellbio/documents/imagej_colorsegmentpdf.pdf

Image.. Plugins..threshold color...move saturation slider to select
ROI..close plugin..image..type...8bits..image..adjust...t hreshold..clt+M.

I know there may be other methods for color segmentation but thats all i
know. I cant use color deconvolution as i cant do background correction
calculation ...i am just a pharmacologist.

Thanks in advance.
Sarah M.Mosaad, M.Sc.

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html