Scmos camera

consisted of four identical water-dipping Olympus UMPLFLN 10×/0.3 objectives, two for illumination and two for detection. Two Toptica iBeam smart lasers were externally triggered for alternating double-sided illumination. The laser beam was split 50/50 and directed onto a continuous running galvanometric mirror (1 kHz, EOPC), which pivoted the light sheet and reduced shadowing effects in the excitation paths due to absorption of the specimen^{37 (link),38} . Light sheets were generated with cylindrical lenses and projected with telescopes and the illumination objectives onto the focal plane of both detection lenses. The focal planes of the two detection objectives were imaged onto two Andor Zyla sCMOS cameras. The whole embryo was imaged from several angles with a z-stack spacing of 2 μm every 30 s up to 5 min for up to 36 h. A custom LabVIEW (National Instruments) program was implemented to adjust stage positions, stack coordinates and various parameters for time-lapse acquisition. A custom fusion program was used for visualization and generation of maximum intensity projections⁴² .
For long-term time-lapse acquisition, embryos were embedded in 0.1% low-melting-point agarose inside fluorinated ethylene propylene (FEP) tubes as described in^{43 (link)–45} . 0.016% tricaine was used in the E3-filled-imaging chamber only when α-bungarotoxin mRNA was not co-injected with DNA constructs.

Imaging was performed on a Zeiss LSM 880 confocal microscope [equipped with a unique scan head incorporating a high-resolution galvo scanner along with two photomultiplier tubes (PMTs) and a 32-element spectral detector as well as a transmitted light PMT for differential interference contrast imaging] or a Nikon spinning disk confocal microscope (equipped with a Yokagawa CSU-X1 variable speed Nipkow spinning disk scan head, Andor Zyla sCMOS cameras, and a light-emitting diode–based DMD (Deformable Mirror Device) system for ultrafast photostimulation). Images were taken using a 10× objective for micropatterns with a single protein and using a 60× or 100× oil-immersive objective for other images.

Cell images of fluorescently labelled cells were obtained using an upright microscope (Nikon eclipse Ni-U) with a water immersion objective ( × 60 magnification, NA=1.0) and an Orca Flash 4.0 camera (Hamamatsu). To obtain three-dimensional stacks, an inverted microscope (Nikon Eclipse Ti) with a spinning disk confocal unit (CSU-W1, Yokogawa), a Zyla sCMOS camera (Andor) and a × 60 objective was used. This objective was either of oil immersion (NA=1.42) or of water immersion (NA=1.0) for experiments involving stretch, as viscous oil droplets dragged the flexible PDMS membrane used for stretch and precluded proper focusing.

A dried film of polystyrene or silica microspheres with various diameters (Polysciences) was formed on cover glass and was then placed on the sample stage of an inverted microscope (OLYMPUS IX71) with the microsphere film side oriented downwards (Fig. S1). A dry dark-field condenser (U-DCD: NA 0.8-0.92) with a 405 nm LED or a halogen lamp as a light source and a long-working-distance 60x objective (NA 0.7) were used for the transmission DF mode. A patterned stop with three symmetric open apertures, each of which span an angle of 60°, was added to the U-DCD condenser for the partial DF illumination. The images were captured using a sCMOS camera (Andor, NEO).

Cells stably-expressing the corresponding GFP- or mCherry-tagged proteins were seeded on 24-wells glass bottom plates (Cellvis) and transfected with the corresponding siRNA. Cells were imaged 48 hours later using 100x oil-immersion objective equipped Nikon Eclipse Ti-E inverted CSU-X1 spinning disk confocal microscope with attached environmental chamber. Images were acquired every 15 seconds for HCT116 cells or every 10 seconds for Hela cells using an Andor Neo sCMOS camera using 2x2 binning. The quantification of the time-lapse images was performed using Fiji (Schindelin et al., 2012 (link)). Briefly, GFP fluorescence around chromatin was selected by manually adjusting the threshold of the green channel. Then a binary mask was created and the total intensity (mean intensity x mask area) of GFP-tagged proteins was quantified for every time point. The curve corresponding to the recruitment of GFP-tagged proteins was represented as mean ± SEM and the beginning of the furrow ingression was set as t0. The area under the curves was quantified using Prism (GraphPad Software).
To compare the timing of GFP-CC2D1B versus CHMP4B-L-GFP recruitment, the first (Ti) and the last (Tf) frames showing GFP accumulation in the perinuclear area were manually scored in every movie and the data were represented as 5-95 percentile boxes using Prism (GraphPad Software).