Hi all (and @tinevez),
I’m trying to analyze the orientation of neuron fibers (see image below), and I think the Directionality plugin for Fiji should be able to do it, but I’m having a couple of difficulties. In the image below, it looks to me like the fibers tend to travel left-right more than up-down, and I’d like to quantify this and have an objective way of measuring this.
In Fourier mode, the analysis (judging by the Orientation map) seems to ignore a lot of the image – many fibers show no color, and the ones that do are clustered near the middle.
This is much less a problem in Local gradient mode, but it does still seem to ignore 1) the dimmer fibers, and 2) regions in the middle of thick fibers. The image below shows a cropped region of the orientation map output, where there is no color on the up-down smaller fibers, and only the edges of the thick fiber are colored.
With regards to the dimmer fibers, can you please tell me how the analysis decides what is real signal and what is background? And do you know of a way that I could dictate what the low threshold is so that real signal is not excluded?
With regards to the thickness of fibers, you might notice that the left-right fibers are typically much thicker than up-down fibers, but the orientation map keeps much of the middle of the tracts un-colored. In reality the thickness represents many fibers bunched together. Do you know of a way to get the analysis to include the full fiber width? The issue here is not resolution - even with very high resolution images at 63x on a confocal, these fibers are too close together to be resolved.
Thanks very much for any help!