Unexpected values in sholl analysis

Hi all,

I’ve got a problem when doing the sholl analysis. The problem is that when I get the results (the crossings at diferent distances), there are some few values that can’t be true. As an example, I’ve got a neuron with 5 branches (paths) and at, for example, 16.2 microns, the Sholl says it’s got 7 crossings (and there is no way you can count 7 branches). It only happens sometimes, and does not follow any pattern.

Thank you in advance!

Welcome to the forum, @Sergi!

Can you post an example image were this issue occurs?

Thank you for the fast answer!

That screenshot is informative, but to reproduce the issue, it would be helpful to also have the original image. Did you threshold the image? How did you get the skeleton lines? Which parameters did you use?

I have got a time-lapse for which I select the point at which the neuron is more stable (in this case I stopped the image at the 166 frame). Then I select the neuron with the rectangular selector and press shift+ctr+D to make a duplicate of the image, and press shft+ctrl+I to invert it. Then I change the brighness/contrast with the auto option, change the image to 8-bit and run the Segmentation->Simple Neurite Tracer plugin. Then I click in the center of the neuron and click again at the end of the neurite, I select the path that I’ve just created and with the ctrl pressed I click again in the center of the image (so the begining of this new path) and at the end of the neurite again, and so on for the diferent branches. Then I select the first path I completed and press shft+ctr+a to make the analysis, I select to use the whole pathes and the circle separation of 0.2.

I tried to upload the time-lapse but as I am new user in the forum it says I can’t.

Hi @Sergi,

I’m guessing you are using Simple Neurite Tracer (SNT). You mention a time-lapse, and the first thing that comes to mind is that SNT was not really designed to deal with images with a time dimension.

It would be indeed useful to have access to your analysis. Can you make the original image and the .traces file accessible through a file sharing service, like wetransfer or dropbox? The shft+ctr+a trigger in Simple Neurite Tracer is not macro recordable, so please report the coordinates (world and image) that you are choosing for focus of the Sholl profile (they are reported in ImageJ status bar).

Yes, as SNT can’t lead with images with time dimension, that’s why I make a duplicate of the image at the time that is more stable to be analysed.

I give you the link of the wetransfer with the time-lapse, as the image I’ve saved is the one already tuned (so I’ve changed the brighness and so). The frame at which I’ve duplicated the image, so the image I use, is 166. And the world and image value, you mean when I press sft+ctrl+a?

Yes, these are the path coordinates reported in the IJ status bar. This will tell us where the Sholl profile is centered.

@Sergi, you forgot to include the .traces file containing the tracings of your neuron at frame t=166. Without it, there is not much we can do to understand the problem.

I upload the traces:

Traces Fiji Forum.zip (10.9 KB)

And here the world and iamge:

@Sergi, it is all very unfortunate, but the zip you uploaded seems to be corrupt. I can only download an empty file. Could you please try an alternative, perhaps the same strategy you used for the czi file?

Here it is, I expect everything goes fine now:

Plus, I also upload it without compressing it:

@Sergi,

It has been rather convolved trying to look into this in an effective way. You shared the traces of the problematic cell as an SNT exported .swc file when we asked for and needed the .traces file. The problem here is that SWC files do not contain any metadata. It is for instance, quite cumbersome to translate those center coordinates you reported into the swc space. Your cell is rather simple and you may thing these requests are overzealousness, but the time we spend guessing what may be wrong, is actually time we could spend looking into what may be actually wrong.

Anyway, you should consider the possibility that one of the neurite extends tangential or quasi-tangential to the sampling circle. In those cases, and specially in a discrete matrix of pixels, a single process can contribute to multiple intersections at the intersection point.
This seems to be an actual possibility:

(BTW, This is actually, one of the reasons why the Sholl plugin implemented multiple samples per radius). It could also be that some rounding errors in the source code contribute to the exacerbated counts you find, made obvious by the simplicity of your cell, and the fact that you seem to be using a radius increment larger than your pixel size.

If you want us to look further, please ensure the issue exists in an updated Fiji installation subscribed to the Java8 update site, have a look at Bug_reporting_best_practices, then include in the shared zip file, the actual image used for tracing, the .traces file, and any other information that may be relevant.