In general, a way to count objects in a 3D image is to use ImageJ's thresholding tools to segment the objects of interest, and then use the "Analyze Particles" function to count the number of objects. Although, I don't know if it will deal with exactly 26 neighbours.

Regarding 3D object counter plugin, I haven't tried it, but in theory it should work. You can follow these steps: Open your 3D image, Go to the "Plugins" menu and select "3D Object Counter" and set the connectivity to "26, click ok and proceed, the plugin ideally should display the number of objects in the image.

Regarding the thresholding function included in the plugin, if you are using a binary image, i don't think that the plugin will perform any additional thresholding, but most likely use the binary values of the image to determine which voxels belong to an object. It would be important to make sure that your binary image is correctly thresholded and only consists of two values (0 and 1), otherwise the plugin may not work correctly.

Svenja Roosch As you mentioned, the 3D Objects Counter plugin in ImageJ uses a 13-neighborhood rather than a 26-neighborhood. When a new object's pixel is identified, it examines the nine pixels on the higher slice and four pixels on the same slice for an existing tag.

This plugin can handle binary pictures, however, because the image is already binary, you must define the thresholding function to include all pixels (min = 0, max = 255).

Another approach is to use the 3D ImageJ Suite, which is a set of plugins for ImageJ that enable 3D image processing. The 3D ImageJ Suite offers the "3D Connected Components" plugin, which employs a 26-neighborhood and can also be used to count items in a 3D binary image stack.

It's vital to remember that ImageJ's Analyze Particles function is meant for 2D analysis and cannot count objects in 3D using the 26-neighborhood.

Dear Svenja Roosch

Class to the plugins folder, or subfolder, restart ImageJ, and there will be a new " 3D objects counter" command in the Plugins menu, or submenu. Description: This plug-in counts the number of 3D objects in a stack and displays the volume, the surface, the centre of mass and the centre of intensity for each object. https://imagej.nih.gov

Regards,

Shafagat

Hello everyone,

thanks to everyone who gave answers so far. I simultaneously contacted one of the developers, Fabrice Cordelières, and he corrected my assumption:

"3D-OC is looking at all the 26 neighbors: 8 in the sample slice, 9 above and 9 below, once the full process has been done. But as the tagging takes place from the top left corner to the bottom right corner, only the 13 previous pixels may have been tagged: this is why I’ve phrased it so."

The point is, while the algorithm is running, it can only consider the 13 voxels of the 26-neighbourhood that have been tagged before but in effect, objects that are connected with respect to a 26-neighbourhood will be tagged as the same object. A colourful drawing might have been helpful to understand that. ^^

So actually, given that the plugin works with binary images, as Qamar Ul Islam and Aditya Kumar suggested, the 3D Objects Counter is exactly what I need.

Btw, I had the impression that two answers ignored a part of the information I gave in my post. Please remember to read carefully! This will increase the value of your answers.

Kind regards,

Svenja

The 3D Objects Counter plugin uses a 26-connectivity criterion, which means that it checks all 26 voxels (pixels in 3D) surrounding a voxel that is currently being processed. The phrase "13 previous neighbors (9 on the upper slice and 4 on the same slice) are checked for an existing tag" refers to the fact that, in addition to the 26 voxels surrounding the current voxel, the plugin also checks for previously processed voxels that are connected to the current voxel. These previously processed voxels are used to assign a label or "tag" to the current voxel, indicating that it is part of the same object.

Regarding the thresholding function, the plugin can handle binary images, however, it is not necessary to use it if you are working with binary images. You can simply set the threshold to 0 and all non-zero voxels will be considered as objects.

Other options to count objects in 3D using ImageJ are using the plugin "3D Object Counter (3D OC)" which is based on the 26-neighborhood connectivity criterion, or using the "Watershed 3D" plugin which is based on the distance transform and the markers concept. Both of the above-mentioned plugin are well-documented. It's always recommended to test different methods and compare the results to see which one works best for your specific dataset.

Svenja Roosch I'm delighted to hear that the 3D Objects Counter plugin meets your needs. When running, the method only evaluates the 13 voxels that have already been tagged, however, objects that are related with regard to a 26-neighborhood will be labeled as the same item.