Re: threshold

[Sarah Mohamed]

Dear Jacqui,
I would like to ask you if i wont be able to do background correction as explained by Gabriel Landini How to correct background illumination in brightfield microscopy [ImageJ Documentation Wiki] (as i dont understand the calculation part so i wont be able to do color deconvolution method),
can i measure area fraction of brown color by splitting color into R,B&G then use G for threshold, is this method valid?
Or this method
www.med.upenn.edu/cellbio/documents/imagej_colorsegmentpdf.pdf
I find that when using threshold method image>adjust>threshold the results may change by a slight movement in sliders of hue & brightness, i may analyse an image and obtain a result and analyse it again after few days and obtain another result with change 10-15%. I am just trying to find a method that gives consistent result.
Thanks in advance.
Regards,
Sarah
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Monday, 20 October 2014, 04:46AM +0200 from Jacqui Ross <[hidden email]>:
Hi Sarah,
You can still quantify DAB staining but you need to measure parameters such as cell number, area, area fraction, distribution pattern, etc. Often area fraction can give you the data you are looking for.
You just shouldn't measure intensities. One of the issues not mentioned in the previous discussion that I referred you to, is the need to set up the microscope perfectly every time for Koehler illumination, lamp temperature, etc. just to get consistent images. A lot of people don't do this correctly.
If I was measuring absolute differences in protein expression, I'd probably be using biochemical techniques, e.g. Western blot.
For relative differences in intensity, you could use immunohistochemistry with a fluorescent tag. However, there are many difficulties along that path as well, e.g. photobleaching, etc. and you need to select your antigen target carefully.
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit 
School of Medical Sciences 
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND
Tel: [phone]
Fax: [phone]
http://www.fmhs.auckland.ac.nz/sms/biru/
-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Sarah Mohamed
Sent: Sunday, 19 October 2014 7:19 a.m.
To:  [hidden email]
Subject: Re: threshold
Dear Jacqui,
thanks for your reply, i appreciate it.
In your opinion, which stain can be used (to avoid the DAB issue with quantification) to quantify a marker in treated and untreated samples?
Kind Regards,
Sarah M. Mosaad, M.Sc.
Department of Pharmacology & Toxicology, School of Pharmacy, Suez Canal University, Egypt.
On Sunday, October 12, 2014 10:06 PM, Jacqui Ross < [hidden email] > wrote:
 
Hi Sarah,
Just noticed that no one seems to have replied to your question. This is probably because we aren't sure what "results" you are referring to and what you mean by "threshold".
If you are measuring areas, then it's OK to use some processing to make the segmentation easier as long as you don't start to bring up some areas which were not really stained.
If you were measuring intensity for fluorescence images, then you shouldn't use any processing which changes the pixel values.
However, we know that measuring intensity/density of DAB staining is not acceptable. This has been discussed many times on the listserver.
Please read the previous thread here:   http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html
<  http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html > in particular the comments by Gabriel Landini in reference to DAB quantification.
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND
Tel: [phone]
Fax: [phone]
http://www.fmhs.auckland.ac.nz/sms/biru/
-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Sarah Mohamed
Sent: Tuesday, 16 September 2014 10:15 p.m.
To: [hidden email]<mailto: [hidden email] >
Subject: threshold
Hi,
Can I manipulate with the image brightness before analysis using threshold?
I am trying to determine brown area stained with DAB using image J, i tried to use threshold and color deconvolution. my images a dark can i use photoshop to adjust background or adjust brightness (using levels or curves) ? or that will change the results?
Regards,Sarah M. Mosaad, M.Sc.
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