Hi everyone
My goal is to create a heatmap ratiometric representation of 2 channels (ch2/ch1);
much like this post here:"3D" pseudocolor for ratiometric images (FRET)
and my raw images are in z-stack.
pls refer to this shareable link for my raw images and final output of the image calculator divide
so I have tried the following steps using 3D image suite plugin ;
1. Suppress the noise; i do this for ch1 and ch2 separately
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Select original stack
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Apply 3D median filter to suppress noise:
1. Plugins->3D->3D Fast Filters
2. then select Median as Filter -
Rename newly created image to Filtered
2. Create background image; again i do this indiv for ch1 and ch2 -
Select original stack
-
Apply 3D median filter to suppress noise:
1. Plugins->3D->3D Fast Filters
2. Select Median as Filter
3. Select 10 pixels Kernel_XY and 3 pixels Kernel_Z -
Rename newly created image to Background
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Create background subtracted denoised image of each channel:
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Process->Image Calculator
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Image1 = Filtered ; Image2 = Background ; Operation = Subtract ;
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Select Create new window and 32-bit result options
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Rename newly created image to Result Ch1 ; or Result Ch2
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calculate ratiometric image
Process->Image Calculator
Image1 = Result Ch2 ; Image2 = Result Ch1 ; Operation = Divide
but my final image (entitled result of divide…tif) is looking very weird.
I expected my background to be black?
May I know what I have missed out??
a screenshot of final image is below(but tif version is in shareable link):