Quantification and morphometric assessment of pseudo-WPBs
Image restoration and 3-dimensional (3D) analysis of pilled-up z-stack
images (least eight fields counting a minimum of 100 cells expressing
VWF for each wt and mutants) were executed by the arivis Vision4D 3.2
software (arivis AG, Imaging Science, Germany). The cells were
highlighted based on edge detection algorithms followed by a smoothing
preprocessing using Arivis’s proprietary algorithm Blob Finder. The
cells expressing VWF (green signal) were elected. Subsequently, the
pseudo-WPBs were detected on the green signal after applying enhancement
preprocessing tasks. The average number of pseudo-WPBs per cell was
calculated by dividing the global count of the WPBs in each acquired
image arena by the cells encompassing at least one WPB. The pseudo-WPBs
features including, length, depth, and width (provided by measuring the
longest, middle, and shortest sides of the 3D oriented bounds,
respectively) as well as the shape (characterized by Sphericity factor,
Ψ) and mean intensities, were computed for whole WPBs-like organelles
population, including 1105 of pseudo-WPBs for wt and between 237-355 for
heterozygous variants. Sphericity (Ψ) describes the roundness of the 3D
segment represented as a value between 0 and 1, where 1 is an ideal
sphere. Mean intensities show the average intensity signal value for
every single pseudo-WPBs.