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Quantification of red in bright field image

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Louise Grant

Jul 17, 2013; 10:27am

Quantification of red in bright field image

Hello,

I`d like to quantify the amount of red in this histo slide using ImageJ. I
am not experience with microscopes and I´m not sure where to start. I
searched the ImageJ archives but everything seems to be for fluorescent
images and not brightfield.

Any help would be great!

Thanks

Louise

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0H3V 0XD 13_4_4 x20 20thJune13 LG 3 (2).jpg (609K) Download Attachment

Kenton Arkill

Jul 17, 2013; 12:50pm

Re: Quantification of red in bright field image

Hi
These are always difficult to quantify. They are very rarely linear to colour (i.e. double brightness = double substance stained for) but if you want the area covered then:

the images are the light source colour minus the tissue absorption (assume = though colours) minus the stain. Where you see red is actually less blue and green.
If you go images/split colour you'll get the three colours separately. then Process/ image calculator and divide the red images by the blue one. You can try to then use the threshold tool to find where is red.
You'll notice that even though it does not look it your image is not evenly illuminated which makes this harder. There are some dark red blotches in the original, if you are after those it works very well and they are nicely bright.

Somebody might have more experience of this than me though.
Kenton

On 17 Jul 2013, at 11:27, Louise Grant wrote:

> Hello,
>
>
>
> I`d like to quantify the amount of red in this histo slide using ImageJ. I
> am not experience with microscopes and I´m not sure where to start. I
> searched the ImageJ archives but everything seems to be for fluorescent
> images and not brightfield.
>
>
>
> Any help would be great!
>
>
>
> Thanks
>
>
>
> Louise
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> <0H3V 0XD 13_4_4 x20 20thJune13 LG 3 (2).jpg>

... [show rest of quote]

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

Cameron Nowell-2

Jul 17, 2013; 12:57pm

Re: Quantification of red in bright field image

In reply to this post by Louise Grant

Hi Louise,

It can be done easily enough. I use the Fiji distribution of ImageJ (www.fiji.sc) so i am not sure if the functionality is available in the standard imagej package but the following worked for me

1. go to Image --> colour --> colour deconvolution
2. choose H AEC as te vector and press OK
3. you should get three images. Select the red channel one (Colour_2)
4. go to image adjust threshold, Make sure dark background is unselected. Adjust the threshold (or the MaxEntropy auto setting seemed to work ok).
5. Measure the image (Analyze --> Measure)

If you want a macro for doing this let me know

Cheers

Cam

Cameron J. Nowell
Research Facilities Manager

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

Email: [hidden email]
Mobile: +61 422882700
Office: +61 9903 9587

LinkedIn: Profile
Research Gate: Profile

________________________________________
From: ImageJ Interest Group [[hidden email]] on behalf of Louise Grant [[hidden email]]
Sent: Wednesday, July 17, 2013 8:27 PM
To: [hidden email]
Subject: Quantification of red in bright field image

Hello,

I`d like to quantify the amount of red in this histo slide using ImageJ. I
am not experience with microscopes and I´m not sure where to start. I
searched the ImageJ archives but everything seems to be for fluorescent
images and not brightfield.

Any help would be great!

Thanks

Louise

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

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

karo03

Jul 17, 2013; 2:07pm

Re: Quantification of red in bright field image

To measure the "amount" of red it might be good to estimate from the segmented/thresholded region the total extinction, that is the integrated density of the extinction of red image, Extinction = -log(Transmission red / 255). The so called IOD (integrated optical density) represents the amount of absorbing material!

Regards
Karsten

Am 17.07.2013 um 14:57 schrieb Cameron Nowell <[hidden email]>:

> Hi Louise,
>
> It can be done easily enough. I use the Fiji distribution of ImageJ (www.fiji.sc) so i am not sure if the functionality is available in the standard imagej package but the following worked for me
>
> 1. go to Image --> colour --> colour deconvolution
> 2. choose H AEC as te vector and press OK
> 3. you should get three images. Select the red channel one (Colour_2)
> 4. go to image adjust threshold, Make sure dark background is unselected. Adjust the threshold (or the MaxEntropy auto setting seemed to work ok).
> 5. Measure the image (Analyze --> Measure)
>
>
> If you want a macro for doing this let me know
>
>
> Cheers
>
> Cam
>
>
> Cameron J. Nowell
> Research Facilities Manager
>
> Monash Institute of Pharmaceutical Sciences
> Monash University
> 399 Royal Parade
> (Mail address: 381 Royal Parade)
> Parkville, VIC, 3052
> Australia
>
> Email: [hidden email]
> Mobile: +61 422882700
> Office: +61 9903 9587
>
> LinkedIn: Profile
> Research Gate: Profile
>
>
>
>
> ________________________________________
> From: ImageJ Interest Group [[hidden email]] on behalf of Louise Grant [[hidden email]]
> Sent: Wednesday, July 17, 2013 8:27 PM
> To: [hidden email]
> Subject: Quantification of red in bright field image
>
> Hello,
>
>
>
> I`d like to quantify the amount of red in this histo slide using ImageJ. I
> am not experience with microscopes and I´m not sure where to start. I
> searched the ImageJ archives but everything seems to be for fluorescent
> images and not brightfield.
>
>
>
> Any help would be great!
>
>
>
> Thanks
>
>
>
> Louise
>
>
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

... [show rest of quote]

Karsten
[hidden email]

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Cameron Nowell-2

Jul 17, 2013; 10:12pm

Re: Quantification of red in bright field image

I would only stick to area coverage. IOD, intensity etc on chromogenic stains can be varied. They are very rarely stoichiometric and changes in intensity can just be due to slightly longer incubation times.

Cheers

Cam

________________________________________
From: ImageJ Interest Group [[hidden email]] on behalf of Karsten [[hidden email]]
Sent: Thursday, July 18, 2013 12:07 AM
To: [hidden email]
Subject: Re: Quantification of red in bright field image

To measure the "amount" of red it might be good to estimate from the segmented/thresholded region the total extinction, that is the integrated density of the extinction of red image, Extinction = -log(Transmission red / 255). The so called IOD (integrated optical density) represents the amount of absorbing material!

Regards
Karsten

Am 17.07.2013 um 14:57 schrieb Cameron Nowell <[hidden email]>:

... [show rest of quote]

Karsten
[hidden email]

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

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