SpinX’s refined algorithm for recording pole positions is more reliable compared to its previous iteration. (a) Representative maximum projection images of the mitotic spindle in a HeLa mCherry-Tubulin cell showing pole positions as recorded from SpinX’s old (top) and refined (bottom) algorithm. Black circles correspond to the poles recorded by manual analysis, while orange circles correspond to the poles recorded by SpinX. Pole alignment was kept consistent throughout measurements i.e., red-filled circles correspond to pole 1, whereas blue-filled circles correspond to pole 2. T corresponds to the time at which the image was taken. Scale bar = 10 μm. (b) Representative traces of the x-y coordinates of the poles of a single cell across time as recorded from manual analysis, and SpinX’s old and refined algorithm (Pole 1—top; Pole 2—bottom.). (c) Box plots quantifying the absolute coordinate (x-y) difference between manual analysis and either refined (left) or old (right) SpinX for both Poles 1 and 2. N = 4 cells, each cell consisting of 21 measurements for each pole. Kruskal–Wallis H test + post-hoc Dunn’s test with Bonferroni adjustment: *P <0.05 Pole 1 refined vs. Pole 1 old. (d) Box plots quantifying the % difference in spindle pole total displacement between manual analysis and either the refined (left; N = 3 cells) or old (right; N = 4 cells) versions of SpinX. The comparison between manual analysis and SpinX was performed based on x-y coordinates alone, but it should be noted that SpinX performs 3D reconstruction, thus predicting x-y positions while taking into consideration the z-position.