Help needed with method of background substraction

[khanas]

Hi all,

Have come across an image processing issue this morning and would really
appreciate your help! Forgive my ignorance please I am quite new to
microscopy/imageJ!

Essentially to quantify nanoparticle uptake into cells we take images with a
whole cell fluorescent stain and a reflectance channel - the nanoparticles
turn up in the reflectance channel and we use the staining to draw around a
cell and analyse it for mean intensity.

To remove background reflectance other people in my lab have first split the
channels of the image, and then using the max and min controls in B&C
changed the min value until any background has been removed. After this they
have selected the area in question (using the aforementioned stain), and
then Measured mean intensity.

Unfortunately for some reason, potentially due to an update, it now doesn't
measure between the values we set. it only ever gives mean intensity of the
unedited/adulterated image and I have verified this by measuring the same
area a few times with different Max and Min settings.

If I save the file as another format this can work but such a compression
would be a problem from a quantification point of view!

A colleague just showed me this forum and I will have a look through to see if there are any existing solutions but any and all help would be very much appreciated!

Many thanks
Abs

Any help would be very much appreciated!

Thanks

[Krs5]

Dear Abs,

Try if when you select under Analyze > Set Measurements the option 'Limit to threshold' your measurements are as expected.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
University of Leicester
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif

ImageJ workhops 15 and 16 December

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of khanas
Sent: 30 October 2014 15:32
To: [hidden email]
Subject: Help needed with method of background substraction

Hi all,

Have come across an image processing issue this morning and would really appreciate your help! Forgive my ignorance please I am quite new to microscopy/imageJ!

Essentially to quantify nanoparticle uptake into cells we take images with a whole cell fluorescent stain and a reflectance channel - the nanoparticles turn up in the reflectance channel and we use the staining to draw around a cell and analyse it for mean intensity.

To remove background reflectance other people in my lab have first split the channels of the image, and then using the max and min controls in B&C changed the min value until any background has been removed. After this they have selected the area in question (using the aforementioned stain), and then Measured mean intensity.

Unfortunately for some reason, potentially due to an update, it now doesn't measure between the values we set. it only ever gives mean intensity of the unedited/adulterated image and I have verified this by measuring the same area a few times with different Max and Min settings.

If I save the file as another format this can work but such a compression would be a problem from a quantification point of view!

A colleague just showed me this forum and I will have a look through to see if there are any existing solutions but any and all help would be very much appreciated!

Many thanks
Abs

Any help would be very much appreciated!

Thanks



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[khanas]

Dear Kees, 

Thanks for your reply!

I tried that option last night but with no success unfortunately. I just gave it another go in case I was being daft but no luck. 

I think the problem is that the image is at 16bit, and as that is the case ImageJ won't apply any changes in the LUTs - would you happen to know a way around this?

Thanks again for taking the time to write to me. 

Best

Abs

On Fri, Oct 31, 2014 at 11:23 AM, Straatman, Kees (Dr.) [via ImageJ] <[hidden email]> wrote:

Dear Abs,

Try if when you select under Analyze > Set Measurements the option 'Limit to threshold' your measurements are as expected.

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Advanced Imaging Facility
University of Leicester
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif

ImageJ workhops 15 and 16 December

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of khanas
Sent: 30 October 2014 15:32
To: [hidden email]
Subject: Help needed with method of background substraction

Hi all,

Have come across an image processing issue this morning and would really appreciate your help! Forgive my ignorance please I am quite new to microscopy/imageJ!

Essentially to quantify nanoparticle uptake into cells we take images with a whole cell fluorescent stain and a reflectance channel - the nanoparticles turn up in the reflectance channel and we use the staining to draw around a cell and analyse it for mean intensity.

To remove background reflectance other people in my lab have first split the channels of the image, and then using the max and min controls in B&C changed the min value until any background has been removed. After this they have selected the area in question (using the aforementioned stain), and then Measured mean intensity.

Unfortunately for some reason, potentially due to an update, it now doesn't measure between the values we set. it only ever gives mean intensity of the unedited/adulterated image and I have verified this by measuring the same area a few times with different Max and Min settings.

If I save the file as another format this can work but such a compression would be a problem from a quantification point of view!

A colleague just showed me this forum and I will have a look through to see if there are any existing solutions but any and all help would be very much appreciated!

Many thanks
Abs

Any help would be very much appreciated!

Thanks



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[Djoere Gaublomme]

I think you might use 'Process'-->'Math'--> 'Subtract' and enter the  
value you would otherwise use for your Min settings to remove the  
background.

I would just remark though, that when using signal intensity as an  
output/measurement, you really have to stop and think about your setup  
(is it possible to use the same lighting conditions for all samples  
when taking the pictures? , is it possible to always Subtract the same  
value ?, etc.), otherwise your numbers might be rubbish.

greets

Djoere


Quoting khanas <[hidden email]>:

> Hi all,
>
> Have come across an image processing issue this morning and would really
> appreciate your help! Forgive my ignorance please I am quite new to
> microscopy/imageJ!
>
> Essentially to quantify nanoparticle uptake into cells we take images with a
> whole cell fluorescent stain and a reflectance channel - the nanoparticles
> turn up in the reflectance channel and we use the staining to draw around a
> cell and analyse it for mean intensity.
>
> To remove background reflectance other people in my lab have first split the
> channels of the image, and then using the max and min controls in B&C
> changed the min value until any background has been removed. After this they
> have selected the area in question (using the aforementioned stain), and
> then Measured mean intensity.
>
> Unfortunately for some reason, potentially due to an update, it now doesn't
> measure between the values we set. it only ever gives mean intensity of the
> unedited/adulterated image and I have verified this by measuring the same
> area a few times with different Max and Min settings.
>
> If I save the file as another format this can work but such a compression
> would be a problem from a quantification point of view!
>
> A colleague just showed me this forum and I will have a look through to see
> if there are any existing solutions but any and all help would be very much
> appreciated!
>
> Many thanks
> Abs
>
> Any help would be very much appreciated!
>
> Thanks
>
>
>
> --
> View this message in context:  
> http://imagej.1557.x6.nabble.com/Help-needed-with-method-of-background-substraction-tp5010244.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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

[khanas]

Hi Djoere, 

Thanks for your reply - I will give that a go. 

And this is something we had considered on this end when it comes to analysis. We are looking at reflectance images with signal intensity used as a measure of nanoparticle internalisation (our NPs reflect quite brightly within cells). This measure is to cut out the cell background and hopefully quantify what is left over as our nanoparticle signal within each region of interest. We are trying a few different things to see if these attempts work. Essentially we do everything within 24 well plates and use our controls for this substraction technique to 'threshold' and look at the leftover reflectance. 

Thanks again

Abs

On Wed, Nov 5, 2014 at 3:20 PM, Djoere Gaublomme [via ImageJ] <[hidden email]> wrote:

I think you might use 'Process'-->'Math'--> 'Subtract' and enter the  
value you would otherwise use for your Min settings to remove the  
background.

I would just remark though, that when using signal intensity as an  
output/measurement, you really have to stop and think about your setup  
(is it possible to use the same lighting conditions for all samples  
when taking the pictures? , is it possible to always Subtract the same  
value ?, etc.), otherwise your numbers might be rubbish.

greets

Djoere

Quoting khanas <[hidden email]>:

> Hi all,
>
> Have come across an image processing issue this morning and would really
> appreciate your help! Forgive my ignorance please I am quite new to
> microscopy/imageJ!
>
> Essentially to quantify nanoparticle uptake into cells we take images with a
> whole cell fluorescent stain and a reflectance channel - the nanoparticles
> turn up in the reflectance channel and we use the staining to draw around a
> cell and analyse it for mean intensity.
>
> To remove background reflectance other people in my lab have first split the
> channels of the image, and then using the max and min controls in B&C
> changed the min value until any background has been removed. After this they
> have selected the area in question (using the aforementioned stain), and
> then Measured mean intensity.
>
> Unfortunately for some reason, potentially due to an update, it now doesn't
> measure between the values we set. it only ever gives mean intensity of the
> unedited/adulterated image and I have verified this by measuring the same
> area a few times with different Max and Min settings.
>
> If I save the file as another format this can work but such a compression
> would be a problem from a quantification point of view!
>
> A colleague just showed me this forum and I will have a look through to see
> if there are any existing solutions but any and all help would be very much
> appreciated!
>
> Many thanks
> Abs
>
> Any help would be very much appreciated!
>
> Thanks
>
>
>
> --
> View this message in context:  
> http://imagej.1557.x6.nabble.com/Help-needed-with-method-of-background-substraction-tp5010244.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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