Figure 4.
3D reconstruction and modeling for time-resolved analysis of spindle-cortex interaction changes. (a) (i) Representative time-lapse images of a mitotic cell (left) with the corresponding outlined masks (red dashed line) predicted with SpinX’s AI module. SpinX utilizes z-slices of cell cortex boundaries (red, transparent and inner rings) to reconstruct the 3D shape of the cell via Minimum Volume Enclosing Ellipsoidal fit, MVEE (green, dotted outer rings). (ii) Representative time-lapse images of a spindle with the corresponding outlined masks (red dashed line) predicted with SpinX’s AI module. Merging masks with raw images (burn) removes non-spindle signals. PSF was generated and fitted (blue line) to map intensity fluctuations with changes in axial positions for each pixel (red) belonging to the spindle. To reconstruct the 3D structure of the spindle, MVEE (green) was applied. (iii) The 3D plot shows the complete model. The cell cortex is represented by the polygon mesh in gray with the spindle principal axes, which correspond to spindle height (red), width (green), and length (blue). The large orange and black dots at the ends of the length axis correspond to the spindle’s individual poles, while the smaller dots correspond to the ends of the spindle height (red-filled) and width (green-filled) axes. Ray traces from the spindle poles are represented by dashed gray lines, and their intersection points are marked as dark blue dots on the cortical mesh. The pole-cortex distance is outlined in magenta. (b) Demonstration of pole refinement in SpinX. Spindle pole estimations without and with pole refinements are indicated by red dots and green crosses, respectively. (c) Representative line plots show x and y coordinate changes of a spindle tracked through time, for its poles 1 and 2, measured either manually by a beginner (yellow), expert (blue), or automatically with SpinX (green). (d) Representative max projection time-lapse images of a HeLa cell expressing mCherry-Tubulin. Orange and black dots represent spindle poles 1 and 2, respectively. The cell outline (blue) was extracted from the predicted segmentation mask of cell membrane by SpinX’s AI module. (e) Line plots show individual pole-cortex 3D distance measurements computed from d for pole 1 (orange) and pole 2 (black) without and with 3D 6-point tracking, respectively. Scale bars: 5 and 1 µm for PSF in b.

3D reconstruction and modeling for time-resolved analysis of spindle-cortex interaction changes. (a) (i) Representative time-lapse images of a mitotic cell (left) with the corresponding outlined masks (red dashed line) predicted with SpinX’s AI module. SpinX utilizes z-slices of cell cortex boundaries (red, transparent and inner rings) to reconstruct the 3D shape of the cell via Minimum Volume Enclosing Ellipsoidal fit, MVEE (green, dotted outer rings). (ii) Representative time-lapse images of a spindle with the corresponding outlined masks (red dashed line) predicted with SpinX’s AI module. Merging masks with raw images (burn) removes non-spindle signals. PSF was generated and fitted (blue line) to map intensity fluctuations with changes in axial positions for each pixel (red) belonging to the spindle. To reconstruct the 3D structure of the spindle, MVEE (green) was applied. (iii) The 3D plot shows the complete model. The cell cortex is represented by the polygon mesh in gray with the spindle principal axes, which correspond to spindle height (red), width (green), and length (blue). The large orange and black dots at the ends of the length axis correspond to the spindle’s individual poles, while the smaller dots correspond to the ends of the spindle height (red-filled) and width (green-filled) axes. Ray traces from the spindle poles are represented by dashed gray lines, and their intersection points are marked as dark blue dots on the cortical mesh. The pole-cortex distance is outlined in magenta. (b) Demonstration of pole refinement in SpinX. Spindle pole estimations without and with pole refinements are indicated by red dots and green crosses, respectively. (c) Representative line plots show x and y coordinate changes of a spindle tracked through time, for its poles 1 and 2, measured either manually by a beginner (yellow), expert (blue), or automatically with SpinX (green). (d) Representative max projection time-lapse images of a HeLa cell expressing mCherry-Tubulin. Orange and black dots represent spindle poles 1 and 2, respectively. The cell outline (blue) was extracted from the predicted segmentation mask of cell membrane by SpinX’s AI module. (e) Line plots show individual pole-cortex 3D distance measurements computed from d for pole 1 (orange) and pole 2 (black) without and with 3D 6-point tracking, respectively. Scale bars: 5 and 1 µm for PSF in b.

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