Re: measuring grayscale of objects in a plate

Posted by JDing on Jul 27, 2013; 12:20am
URL: http://imagej.273.s1.nabble.com/measuring-grayscale-of-objects-in-a-plate-tp5004160p5004168.html

Maybe an illustrative example will clear up the confusion:

I begin with my plate with 48 seeds. I threshold it so the image is black and white, and my seeds are black, so I now use analyze particles to move the outlines of the seeds to the ROI manager. Since I want to measure color, and the image I have now is black and white, I now revert to my original colored image. Now I use the measure function on the ROI manager to get my results table, with a measurement of the grayscale for each seed.

Assume well A1 is a circle centered at (50, 50) with radius 20. B1 is a circle is centered at (100, 50), with radius 20, etc. Assume that the seed in well A1 is a circle centered at (50, 60) with radius 5. Assume that the seed in B1 is a circle centered at (100, 40) with radius 5. Assume the seed in C1 is circle centered at (150, 50) with radius 10. My ROI manager labels the seed in B1 as "001-35", its smallest y-coordinate is 35, which is the number 1 smallest y-coordinate among all the seeds. (I believe it names based on smallest y coordinate, though it might be the center.) The seed in C1 is labeled "002-40" and the seed in A1 is labeled "003-55." Then on the results table, it lists "001-35" then "002-40" then "003-55." The labels A1, B1, and C1 do not appear anywhere on the results table, because ImageJ does not know that the well at (50, 50) is called A1. so I have no way of telling that 001-35 in fact comes from B1 unless I go back to the ROI manager and manually click each one to check. That would take too much time. So from here, how should I reorder the rows without having to do it manually?

I can't use my saved ROI set to measure the grayscale of the seeds, since the ROI set has ROIs for the wells. So, for example, I have an ROI labeled "A1" that corresponds with well A1. However, if I measured the color of the ROI "A1" I would not get a correct grayscale for  the seed in A1, because I'm also measuring the part of the well that does not contain the seed. In short, my saved ROI set is useless in this task, and should be ignored, unless it is possible to perform the functions of ImageJ only within the boundaries a certain selected ROI (and not to the entire image), in which case my problem would be solved.

Hopefully this makes clear what my exact problem is.

JDing

On Fri, Jul 26, 2013 at 5:57 PM, Karsten Rodenacker-3 [via ImageJ] <[hidden email]> wrote:

Centroid coordinates of any ROI (1 and 2) are coordinates of your plate image. Sorting in y means that you can group the "lines" and sorting in x allows to group the "columns". Each well covers a box in which the related roi coordinates are falling, if not, than the roi is not inside the well. Where is the problem?

Regards,
Karsten

Is it possible that you think image coordinates of ImageJ are no
Am 27.07.2013 um 00:14 schrieb JDing <[hidden email]>:

> I am confused about what "your ROIs" means. Do you mean 1) the ROI set that
> I have saved, which has one ROI for each of the 48 wells, or 2) the ROIs
> that I get after I threshold/analyze particles/save to ROI manager?
>
> If you mean (1), my saved ROI set has ROIs that includes each well in its
> entirety. However, since seeds take up only a fraction of the well, and
> since seeds can be in many different shapes and sizes and places within the
> well, my ROI set doesn't actually help very much in identifying the seeds
> themselves. In my original post, I suggested that if I could apply the
> threshold/analyze particle functions to only the ROI and not the entire
> image, then I could apply the process 48 times, one time for each ROI/well,
> which would then be in order, since my ROI set is already in order, and I
> do this in the order of my ROI set. However, I do not know how to apply the
> functions to only the ROI, so I can't use this method (unless you know how
> to do it).
>
> If you mean (2), the seeds vary in position within each well. So even
> though in theory, if I tell the program to list in increasing y-coordinate
> values and then in increasing x-coordinate values, I should get the
> wells/seeds in order, this is not the case in practice. This is what I
> tried to explain in my previous post. Unless I am misunderstanding you, in
> which case you would have to explain your method in slightly more detail.
>
> Thanks
>
> JDing
>
>
> On Fri, Jul 26, 2013 at 4:09 PM, Karsten Rodenacker-3 [via ImageJ] <

> [hidden email]> wrote:

>
>> Assuming your wells build a chequerboard and the image is aligned with it,
>> the coordinates of your ROIs will allow the ordering of the results. Of
>> course the actual list of result entries depend on the sequence of your
>> ROIs in the ROI manager rspw. on the first found pixel per region.
>>
>> Possibly you have to load the results into Excel or R for the ordering in
>> x and y.
>>
>> Regards
>> Karsten
>>

>> Am 26.07.2013 um 22:55 schrieb JDing <[hidden email]<http://user/SendEmail.jtp?type=node&node=5004164&i=0>>:

>>

>>
>>> You understand me correctly, but your suggestion doesn't seem to work. I
>>> added "center of mass" to the measurements and it didn't put the wells
>> in
>>> order. Here is my explanation: even though the sides of my plate are
>> aligned
>>> with the x and y axes, the seeds themselves vary in their position
>> within
>>> the well (and their size). So the seed in A1 could be centered at
>> coordinate
>>> (50, 50), while B1 is at (100, 49), and C1 is at (150, 49.5), etc. In
>> this
>>> case, if ImageJ measured/listed in order of increasing y-coordinate (the
>>> default for my computer), it would list B1 and C1 before A1. So this
>> method
>>> doesn't work well in real practice.
>>>
>>> Thank you for your consideration, though.
>>>
>>> JDing
>>>
>>>
>>>
>>> --
>>> View this message in context:
>> http://imagej.1557.x6.nabble.com/measuring-grayscale-of-objects-in-a-plate-tp5004160p5004163.html

>>
>>> Sent from the ImageJ mailing list archive at Nabble.com.
>>>
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>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>
>> Karsten

>> [hidden email] <http://user/SendEmail.jtp?type=node&node=5004164&i=1>

>>
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Karsten
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