percentage of area covered by cells

[dianalves]

Hi everyone,

I am a new user of ImageJ and I would be very grateful for any help with the following problem.
I want to use ImageJ to calculate total percentage of area covered by bacteria and distinguish the fraction of dead and live cells. For that I am using a live or dead staining where live cells are stained in green and dead cells in red. I obtained a single grayscale image for each fluorescence channel and after subtracting background, I set a thereshold value manually for each image.  Afterwards I went to Measure and checked Area and Area fraction (%). I thought that the values of Area fraction was the percentage of area covered but the values seem to low. For instance, the percentage of area covered for the image I have attached (if I consider area fraction) is of about 10%. Can you please let me know if I am doing something wrong or not interpreting the values as I should?  
Thanks in advance for any ideas!

[gankaku]

Hi Diana,

Could you attatch also a example image after your extraction procedure
(binary image after the thresholding)?
Then it might be easier to find the problem or explanation.
When I try to repeat your described method with a quick extraction (while
my thresholding will obviously be different from yours), I end up with
values between 35 or 45% (Area fraction) depending upon the threholding I
apply. But it seems those are realistic values in comparison to what I see
in my image.
A potential problem might be that you background option is not set
correctly. This you can check under >Process >Binary >Options... to figure
out if ImageJ considers the background to be black or white. In conjunction
with this, your output image might be displayed with an inverted LUT (for
whatever reason) which would give you in inverted measurement in comparison
to what you would consider as being foreground (features of interest).
Additionally make sure that you do not have any selection present in the
image. Otherwhise, the area fraction is determined only from within your
ROI.

Hope this helps already. With a binary image posted we might help you
further.
Kind regards,
Jan

2014-11-28 11:49 GMT+01:00 dianalves <[hidden email]>:

> Hi everyone,
>
> I am a new user of ImageJ and I would be very grateful for any help with
> the
> following problem.
> I want to use ImageJ to calculate total percentage of area covered by
> bacteria and distinguish the fraction of dead and live cells. For that I am
> using a live or dead staining where live cells are stained in green and
> dead
> cells in red. I obtained a single grayscale image for each fluorescence
> channel and after subtracting background, I set a thereshold value manually
> for each image.  Afterwards I went to Measure and checked Area and Area
> fraction (%). I thought that the values of Area fraction was the percentage
> of area covered but the values seem to low. For instance, the percentage of
> area covered for the image I have attached (if I consider area fraction) is
> of about 10%. Can you please let me know if I am doing something wrong or
> not interpreting the values as I should?
> Thanks in advance for any ideas!
> <http://imagej.1557.x6.nabble.com/file/n5010672/Live_cells.png>
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/percentage-of-area-covered-by-cells-tp5010672.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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[Joel Sheffield]

Hi,

It seems to me that your image contains two levels of intensity, at least,
This seems to correspond as well to the number of pixels that are present
in each particle. When I enlarge the image so that I can see the individual
spots, I can measure intensities from about 80 to about 200. I wonder if
this is due to a focus artifact.  At any rate, it makes the choice of
threshold particularly sensitive.  If I set the threshold to include mostly
the brightest particles, from 150-255I get a percent area of about 10%, but
it is clear that I am not capturing all of the particles in the the frame.
If I reset the threshold so that I capture all of the visible particles,
i.e. with a setting of about 75-255, I get approximately 50% of the image.

When you slide the threshold bar across the image (in an 8 bit image), you
will see underneath an indication of the percent of the area that you are
including.

It is up to you to determine which setting is ideal for your interest.

Joel



Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  *http://tinyurl.com/khbouft <http://tinyurl.com/khbouft>*

On Fri, Nov 28, 2014 at 5:49 AM, dianalves <[hidden email]> wrote:

> Hi everyone,
>
> I am a new user of ImageJ and I would be very grateful for any help with
> the
> following problem.
> I want to use ImageJ to calculate total percentage of area covered by
> bacteria and distinguish the fraction of dead and live cells. For that I am
> using a live or dead staining where live cells are stained in green and
> dead
> cells in red. I obtained a single grayscale image for each fluorescence
> channel and after subtracting background, I set a thereshold value manually
> for each image.  Afterwards I went to Measure and checked Area and Area
> fraction (%). I thought that the values of Area fraction was the percentage
> of area covered but the values seem to low. For instance, the percentage of
> area covered for the image I have attached (if I consider area fraction) is
> of about 10%. Can you please let me know if I am doing something wrong or
> not interpreting the values as I should?
> Thanks in advance for any ideas!
> <http://imagej.1557.x6.nabble.com/file/n5010672/Live_cells.png>
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/percentage-of-area-covered-by-cells-tp5010672.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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