Appendix 6—table 3. Parameters guide for Segmentation.

Menu C and D in Appendix 6—figure 1.

Process type	Parameter name	Description	Range	Default
Segmentation	Algorithm	Defines which algorithm will be used for segmentation.	menu	‘GASP’
	Save Directory	Create a new sub folder where all results will be stored.	text	‘GASP’
	Under/Over seg. fac.	Define the tendency of the algorithm to under of over segment the stack. Small value bias the result towards under-segmentation and large towards over-segmentation.	(0.0…1.0)	0.6
	Run Watersed in 2D	If True the initial superpixels partion will be computed slice by slice, if False in the whole 3D volume at once. While slice by slice drastically improve the speed and memory consumption, the 3D is more accurate.	bool	True
	CNN Prediction Threshold	Define the threshold used for superpixels extraction and Distance Transform Watershed. It has a crucial role for the watershed seeds extraction and can be used similarly to the ‘Unde/Over segmentation factor’ to bias the final result. An high value translate to less seeds being placed (more under segmentation), while with a low value more seeds are placed (more over segmentation).	(0.0…1.0)	0.5
	Watershed Seeds Sigma	Defines the amount of smoothing applied to the CNN predictions for seeds extraction. If a value of 0.0 used no smoothing is applied.	float	2.0
	Watershed Boundary Sigma	Defines the amount of Gaussian smoothing applied to the CNN predictions for the seeded watershed segmentation. If a value of 0.0 used no smoothing is applied.	float	0.0
	Superpixels Minimum Size	Superpixels smaller than the threshold (voxels) will be merged with a the nearest neighbour segment.	integer	50
	Cell Minimum Size	Cells smaller than the threshold (voxels) will be merged with a the nearest neighbour cell.	integer	50
Segmentation Post-processing	Convert to tiff	If True the segmentation is exported as tiff file.	bool	False