Csu x1 scan head

SWG59 (for mitosis) or SWG16 (for apoptosis) young adult worms were paralysed in 0.1% tetramisole (Sigma-Aldrich T1512) for 3 min on a cover slip precoated with 0.1% poly-l-lysine (Sigma-Aldrich P8920) and mounted on 2% agarose pads. Images were acquired with a spinning-disk confocal microscopy (Zeiss C-Apochromat, ×63/1.2NA, Yokogawa CSU-X1 scan head and Hamamatsu ORCA-Flash4.0 camera) on 41 different z planes spaced 1 μm apart, recording one full stack every 30 s. After maximal projections of individual movies in Fiji, the number of metaphase spindles or each new engulfment event—together with their position relative to the distal tip or proximal turn—were manually measured. The frequency of mitotic or apoptotic events along the total amount of imaging time was then binned to obtain a probability of event per unit length of the germline (Supplementary Fig. 1c). From these rates, an estimate of cumulative probability can be deduced. For example, at position x of the gonad, considering that the rate of apoptosis per hour is p_a, then the associated total probability of a cell to survive a period of 3 h is $p_{\mathrm{s}}={\left(1-p_{\mathrm{a}}\right)}^3$ . Hence, the estimated total cell death should be ~100 × (1 − p_s)%.

Raw data collection was performed as previously described^{7 (link)}. Briefly, young-adult hermaphrodite C.elegans expressing the synaptic vesicle precursor marker RAB-3 in the DA9 motor neuron (wyIs251[Pmig-13::GFP::RAB-3]) were paralyzed in 0.3 mM Levamisole in M9. Once paralyzed, worms were carefully transferred to M9 solution on a 10% agarose pad for imaging. This results in an effective Levamisole concentration which is significantly lower than the concentration that was suggested to affect axonal transport^{42 (link)}. Worms were maintained on the pad for no more than 20 min, although we confirmed that viability was maintained even after 4 h.
Fluorescence imaging was performed using a Nikon 60 × CFI plan Apo VC, NA 1.4 objective on a Nikon Ti-E microscope equipped with Yokogawa CSU-X1 scan-head and a Hamamatsu C9100-50 EM-CCD camera at a frame rate of 110 ms/frame, and 240 nm per pixel.
Post-analysis fluorescence movies were corrected and analyzed in imageJ. Animal movement was corrected using FIJI plugin StackReg. Kymographs were generated with KymoBuider. Intensity was averaged ± 5 pixels transverse to the kymograph line.

L4 mothers were maintained overnight at 20°C before dissection in M9 buffer. For imaging, one-cell embryos were mounted on 2% agar pads, thereby slightly compressed to increase the cortical surface visible in a confocal plane. Images were acquired with spinning disk confocal microscope (Zeiss C-Apochromat, 63X/1.2 NA or 100X/1.42 NA, Yokogawa CSU-X1 scan head and Hamamatsu Orca-Flash4.0 camera) every 2 s on two or three different planes (one or two at the bottom for a cortical section, and one 12 µm above for a medial section of the embryo) . For 3D acquisitions, Z-stacks were acquired every 0.2 μm. We assume the shape to be rotationally symmetrical to the longer axis, thus the outline of one medial section of the embryo reflects faithfully cell shape changes and ingression distance.

Imaging was executed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany). The system consists of a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. Z-sectioning was performed with a Piezo-driven motorized stage (Applied Scientific Instrumentation, Eugene, OR, United States) using a Leica HC PL APO 63X/1,4-0,6 oil objective. All acquisitions were performed at 20–23°C.
For most experiments, we collected z-stacks with 45 steps at 1.0 μm distance each with 2, 3, 4, or 5 min intervals, respectively, for a total duration of 1–3 h for acquiring early lima bean to 1.5-fold stage embryos. For imaging of the head-on-view, we used 3–4 min intervals to avoid tipping of embryos. For lineaging, we performed long-term imaging for 250 time points at 3 min intervals and with z sampling of 1 μm over a distance of 30 μm. For acquiring of embryos for one time point before and a time-lapse series after UV laser ablation, we used 2 min intervals and z sampling at 1 μm over a distance of 40–45 μm.

Appropriately staged embryos were imaged using a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) consisting of a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. Z-sectioning was performed with a Piezo-driven motorized stage (Applied Scientific Instrumentation, Eugene, OR, United States). All acquisitions were performed at 20–23°C using a Leica HC PL APO 63X/1.4-0.6 oil objective. For imaging/quantifying of anterior and posterior contacts of ABpl, we collected z-sections of 16 focal planes (0.5 μm apart) with 10 s intervals with a 488 and 561 nm laser at an exposure of 150 ms from the onset of ABa/ABp division, for a total duration of 10 min. While for PIV analysis, we collected 8 z-sections (0.5 μm apart) with 3 s intervals, again for a total duration of 10 min with the same laser settings as above. For imaging 12-cell stage embryos, we collected 26 z-sections (1 μm apart) with intervals of 1 min. For most imaging, laser intensities used were 5% for the 488 nm and 40 or 60% for the 561 nm laser (each 25 mW maximal output at the source).

UM208 young adult worms (24 h post L4) were paralysed in 0.1% tetramisole (Sigma-Aldrich T1512) for 3 min on a cover slip precoated with 0.1% poly-l-lysine (Sigma-Aldrich P8920) and mounted on 2% agarose pads. Images were acquired with spinning-disk confocal microscopy (Zeiss C-Apochromat, ×63/1.2NA, Yokogawa CSU-X1 scan head and Hamamatsu ORCA-Flash4.0 camera) on 100 different z planes spaced 0.5 μm apart. To capture the full gonads, 3 to 4 fields were acquired and stitched together using Fiji’s pairwise stitching plugin. Curved gonads were straightened using a 300 pixels (32 μm) wide line on the x–y stacks using Fiji’s straighten tool and resliced along the z axis to get the cross-sections along the distal to proximal axis (semi-automatic using ‘sideviews’ Fiji macro). For 1/100th of the cross-section slices, the curvature along all the cell–cell interfaces were calculated by fitting a 5 point B-spline curve using the Kappa plugin in Fiji. The average curvature (μm⁻¹) at each cross-section was then plotted along the distal to proximal axis. A smooth curved surface of a cell of 10.0 μm height and 0.5 μm maximal deflection would correspond to a curvature of ~0.04 μm⁻¹, larger than the estimated curvatures shown in Supplementary Fig. 4b.

Young adult worms (24 h post L4) were paralysed in 0.1% tetramisole (Sigma-Aldrich T1512) for 3 min on a cover slip precoated with 0.1% poly-l-lysine (Sigma-Aldrich P8920) and mounted on 2% agarose pads. Images were acquired with a spinning-disk confocal microscopy (Zeiss C-Apochromat, ×63/1.2NA, Yokogawa CSU-X1 scan head and Hamamatsu ORCA-Flash4.0 camera).