Volocity acquisition software

Sqh-GFP embryos were prepared for live imaging and were imaged using a spinning disk confocal microscope (Ultraview; PerkinElmer) with a 60×/1.4 NA oil-immersion objective (Nikon), a 488-nm laser, and an electron-multiplying charge-coupled device camera (C9100-13; Hamamatsu). The microscope was controlled with Volocity acquisition software (Improvision). Ablation was performed using a Micropoint laser (Andor Technology) tuned to 365 nm. For each ablation, a focused laser beam was targeted to the middle of an edge marked by Sqh-GFP to generate a point incision. Time-lapse movies of a single z-slice focused at the level of the invagination front were acquired immediately before and after ablation to measure the movement of surrounding tissues upon release of tension. As a control, ablation was performed at the apical cortex in embryos at cycle 13 anaphase.
Velocity maps of myosin flow during the flow phase and tissue movement immediately after laser ablations were generated using the MATLAB-based software OpenPIV (Taylor et al., 2010 (link)) with a spacing/overlap of 8×8 pixels and an interrogation window size of 32×32 pixels. For the laser-ablation experiments, average velocity map was generated from 24 ablations in eight embryos at approximately 5 min after the onset of cellularization.

The exposed liver lobe was visualized with an Olympus IX81 inverted microscope equipped with a confocal light path (Wave-Fx; Quorum) based on a modified Yokogawa CSU-X1 head (Yokogawa Electric Corporation) with a UPLANSAPO 10×/0.40 or UPLANSAPO 20×/0.70 air objective. Four laser excitation wavelengths (491, 561, 643, and 730 nm; Cobalt) were used in rapid succession and visualized with the appropriate long-pass filters (Semrock). Exposure times for excitation wavelengths were 400 ms for all lasers. A back-thinned EMCCD 512×512 pixel camera (C9100–13, Hamamatsu, Bridgewater, NJ) was used for fluorescence detection. Volocity acquisition software (Improvision) was used to drive the microscope.

Images were acquired with an UltraVIEW spinning disk confocal microscope utilizing a Hamamatsu Flash 4.0 v.2 sCMOS camera. A 40×, NA 1.3 Zeiss EC Plan-Neofluar oil objective was used for all tiled images, and a 63×, NA 1.4 Zeiss Plan-Apochromat oil objective was used for all other images. Imaging was controlled by Volocity acquisition software (PerkinElmer, Waltham, MA). Image stacks were recorded at 350-nm intervals. Tiled images were stitched using Volocity. Six images were acquired of each tissue section, and a total of four animals were used per group per time point. The PLA signal-imaging frame was quantified using Volocity software. The PLA puncta were identified as objects with a signal above a minimum threshold. The sum of PLA signal volume was measured for each frame. Two different statistical methods were used, depending on the number of time points in the experiment. For single-time point experiments, a one-way Kruskal-Wallis ANOVA test was used with a Dunn’s multiple comparisons test. For multiple-time point experiments, a two-way ANOVA was used with a Tukey’s multiple comparisons test. Statistics were performed in GraphPad Prism 7.0.

In vitro images were taken using a Hamamatsu Flash 4.0 version (v.)2 sCMOS camera on a PerkinElmer UltraView spinning-disk confocal microscope on a Zeiss Axiovert 200 M body. A 63×, numerical aperture (NA) 1.4 Zeiss Plan-Apochromat oil objective was used for all images. Imaging was controlled by Volocity acquisition software (PerkinElmer, Waltham, MA). Image stacks were recorded at 200-nm intervals.

Images were acquired with a Hamamatsu Flash 4.0 v2 sCMOS camera on a PerkinElmer UltraView spinning disk confocal microscope mounted to a Zeiss Axiovert 200 M body with a x63 NA 1.4 plan-apochromat objective. Images were acquired with Volocity Acquisition Software (PerkinElmer) with z-stack intervals of 200 nm. Images were linearly contrast enhanced for visual clarity. Images were uniformly contrast enhanced across an experiment.