Posted by
ekamis on
Mar 04, 2021; 7:41am
URL: http://imagej.273.s1.nabble.com/Segmentation-of-objects-when-the-signal-intensity-varies-among-them-tp5024496.html
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http://imagej.1557.x6.nabble.com/file/t382821/Example_bacteria_FM4-64.jpg>
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http://imagej.1557.x6.nabble.com/file/t382821/Example_bacteria_FM4-64_BINARY.jpg>
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http://imagej.1557.x6.nabble.com/file/t382821/Example_bacteria_FM4-64_CLEAR.jpg>
Hi,
I am quite new to Fiji and have encountered a following challenge.
I have a set of images of bacteria taken using 2 channels, DAPI (for DNA)
and FM-64 (for cellular membrane). My goal is to do the segmentation so that
I get a set of individual ROIs (one for each bacterium) based on DNA and
another set of individual ROIs based on the membrane. And then I want to
measure the features of the objects (bacteria) in the original image
surrounded by ROIs.
I constructed a macro that worked OKish for the DAPI channel but not for the
FM4-64 channel. My problem is that I don’t manage to threshold all objects
of interest (all bacteria) in one operation. When I try to apply a
thresholding algorithm from the available list, some bacteria still remain
below the threshold. I assume this is because the FM 4-64 signal is much
stronger in some cells then in the others (see picture Example bacteria
FM4-64).
I tied to apply Filters (I liked Median the most) and Background Subtraction
(radius 10, comparable to the size of my objects) before the thresholding
and it helped a bit, but still the pale cells were left as a background.
So what I did was that I ran a 2-step segmantation, the first step for the
“bright” cells and the second for the “pale” cells. I am not sure if this an
acceptable way to do it. Because I had to apply one thresholding algorithm
at the first step, then after the measurements I just cleared the selected
ROIs in the original image (which created some “black holes” in it, see
picture Example bacteria FM4-64 CLEAR) . And ran another thresholding
algorythm (a different one!) on the remaining objects…
Another thing I tried was to use a Filter called Unsharp Mask or a CLAHE
(Enhance Local Contrast) function, and most cells were identified as objects
and thresholded. But since some of the cells form chains, those chains
appeared as single particles, so I had to apply Watershed which created some
artificial “septa” where they shouldn’t be (see picture Example bacteria
FM4-64 BINARY).
Maybe you could suggest any ideas for a better thresholding? Or
pre-processing before the thresholding? Or other options then Watershed to
“divide” the cells?
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