fluorescent intensity measurement problem

[nurul]

Hi all,

I'm currently facing some problem in using Image J to measure my fluorescence image.
I have already set the ROI and the threshold  that I wanted.
However, when I wanted to measure the image, it didn't measure my ROI according to the threshold that I listed.
May I know the steps in measuring the intensity in the specific ROI. Currently, I'm measuring the intensity in RGB colour as my fluorescence will appear red in a dark background.
If there is any video tutorial, it would be very helpful (as I'm new to this).


Nurul


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

[wirthvolker]

Hi,
from what I understand, I suggest to start by converting your image to
greyscale, 32bit (Image->Type->32bit), then threshold and use
Process->Math->NaN Background to get rid of the pixels below the
threshold. Then you can mesure your ROI and this shold only take the
remaining pixels into account.

run("Blobs (25K)");
run("32-bit");
//run("Threshold...");
setAutoThreshold("Default dark");
run("NaN Background");
makeRectangle(57, 20, 157, 121);
run("Measure");

Best regards,
Volker

2012/9/25 Nurul Alimah Abdul Nasir <[hidden email]>:

> Hi all,
>
> I'm currently facing some problem in using Image J to measure my fluorescence image.
> I have already set the ROI and the threshold  that I wanted.
> However, when I wanted to measure the image, it didn't measure my ROI according to the threshold that I listed.
> May I know the steps in measuring the intensity in the specific ROI. Currently, I'm measuring the intensity in RGB colour as my fluorescence will appear red in a dark background.
> If there is any video tutorial, it would be very helpful (as I'm new to this).
>
>
> Nurul
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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

[Jacqueline Ross]

Hi Nurul,

Are you clicking "Measure" in the ROI Manager? You need to do the measurements from the ROI Manager, otherwise the entire image is measured.

Also, make sure that you have selected "Limit to Threshold" in the "Set Measurements" dialogue box.

What do you mean about your fluorescence appearing red in a dark background? You can make your RGB image grayscale by going to Image - Type - 8bit and then any signal that is detected in the red channel will also be included. This is probably important for you because you are imaging autofluorescence rather than a normal fluorophore.

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 Nurul Alimah Abdul Nasir
Sent: Wednesday, 26 September 2012 1:50 a.m.
To: [hidden email]
Subject: fluorescent intensity measurement problem

Hi all,

I'm currently facing some problem in using Image J to measure my fluorescence image.
I have already set the ROI and the threshold  that I wanted.
However, when I wanted to measure the image, it didn't measure my ROI according to the threshold that I listed.
May I know the steps in measuring the intensity in the specific ROI. Currently, I'm measuring the intensity in RGB colour as my fluorescence will appear red in a dark background.
If there is any video tutorial, it would be very helpful (as I'm new to this).


Nurul


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

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

[nurul]

Thank you all for helping.

I'm trying to adjust my way in measurement.
I have already measure under ROI manager and limit it to threshold but failed to get the result. I may need to change it to 8-bit image though.
Anyway, is there any way to analyze particle that is being selected when thresholding the image only in the ROI?
I've tried to threshold the image( RGB) and then click select, where some particle outline appear. However, some of the particle is not in my ROI. Is it possible to exclude these particle as I'm only interested in those particle inside my ROI.

Thank you.
 
Nurul 


________________________________
 From: Volker Wirth <[hidden email]>
To: [hidden email]
Sent: Wednesday, 26 September 2012 12:01 AM
Subject: Re: fluorescent intensity measurement problem
 
Hi,
from what I understand, I suggest to start by converting your image to
greyscale, 32bit (Image->Type->32bit), then threshold and use
Process->Math->NaN Background to get rid of the pixels below the
threshold. Then you can mesure your ROI and this shold only take the
remaining pixels into account.

run("Blobs (25K)");
run("32-bit");
//run("Threshold...");
setAutoThreshold("Default dark");
run("NaN Background");
makeRectangle(57, 20, 157, 121);
run("Measure");

Best regards,
Volker

2012/9/25 Nurul Alimah Abdul Nasir <[hidden email]>:

> Hi all,
>
> I'm currently facing some problem in using Image J to measure my fluorescence image.
> I have already set the ROI and the threshold  that I wanted.
> However, when I wanted to measure the image, it didn't measure my ROI according to the threshold that I listed.
> May I know the steps in measuring the intensity in the specific ROI. Currently, I'm measuring the intensity in RGB colour as my fluorescence will appear red in a dark background.
> If there is any video tutorial, it would be very helpful (as I'm new to this).
>
>
> Nurul
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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

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

[Jacqueline Ross]

Hi Nurul,

Try clearing the area outside your ROI. Select the ROI and go to "Edit - Clear Outside". Make srue that the background colour swatch (eyedropper tool) is white so that the area cleared is filled with white. If you only have one region, you can then use the Analyze Particles option under the Analyze menu because you don't need the ROI Manager anymore.

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 Nurul Alimah Abdul Nasir
Sent: Wednesday, 26 September 2012 1:36 p.m.
To: [hidden email]
Subject: Re: fluorescent intensity measurement problem



Thank you all for helping.

I'm trying to adjust my way in measurement.
I have already measure under ROI manager and limit it to threshold but failed to get the result. I may need to change it to 8-bit image though.
Anyway, is there any way to analyze particle that is being selected when thresholding the image only in the ROI?
I've tried to threshold the image( RGB) and then click select, where some particle outline appear. However, some of the particle is not in my ROI. Is it possible to exclude these particle as I'm only interested in those particle inside my ROI.

Thank you.
 
Nurul 


________________________________
 From: Volker Wirth <[hidden email]>
To: [hidden email]
Sent: Wednesday, 26 September 2012 12:01 AM
Subject: Re: fluorescent intensity measurement problem
 
Hi,
from what I understand, I suggest to start by converting your image to greyscale, 32bit (Image->Type->32bit), then threshold and use
Process->Math->NaN Background to get rid of the pixels below the
threshold. Then you can mesure your ROI and this shold only take the remaining pixels into account.

run("Blobs (25K)");
run("32-bit");
//run("Threshold...");
setAutoThreshold("Default dark");
run("NaN Background");
makeRectangle(57, 20, 157, 121);
run("Measure");

Best regards,
Volker

2012/9/25 Nurul Alimah Abdul Nasir <[hidden email]>:

> Hi all,
>
> I'm currently facing some problem in using Image J to measure my fluorescence image.
> I have already set the ROI and the threshold  that I wanted.
> However, when I wanted to measure the image, it didn't measure my ROI according to the threshold that I listed.
> May I know the steps in measuring the intensity in the specific ROI. Currently, I'm measuring the intensity in RGB colour as my fluorescence will appear red in a dark background.
> If there is any video tutorial, it would be very helpful (as I'm new to this).
>
>
> Nurul
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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

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

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