Dic 3 prism

Cells were plated on a 24-well μ-Plate (Ibidi®). The medium was changed to CO₂ Independent Medium (Gibco®) 6 h before filming. DNA was stained by addition of the SiR-Hoechst-647 Dye (Spirochrome) to the medium 1 h before imaging. Cells were imaged every 5–10 min in a heated chamber (37 °C) on a 3i Marianas^TM system (Intelligent Imaging Innovations, Inc.) equipped with Axio Observer Z1 microscope (Zeiss), Plan-Apochromat ×40/1.4NA oil objective, M27 with DIC III Prism (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu), and controlled by Slidebook Software 6.0 (Intelligent Imaging Innovations, Inc).

The 2D dataset provided with our notebooks was generated by plating U-251 glioma cells expressing endogenously tagged paxillin- GFP on fibronectin-coated polyacrylamide gels (stiffness 9.6 kPa)^{72 (link)}. Cells were then recorded live using a spinning-disk confocal microscope equipped with a long working distance of ×63 (NA 1.15 water, LD C-Apochromat) objective (Zeiss). The 3D dataset provided with our notebooks was generated by recording A2780 ovarian carcinoma cell, transiently expressing lifeact-RFP (to visualise the actin cytoskeleton), migration on fibroblast-generated cell-derived matrices^{81 (link)}. The cell-derived matrices were labelled using Alexa Fluor 488-recombinant fibronectin and the images acquired using a spinning-disk confocal microscope equipped with a 63x oil (NA 1.4 oil, Plan-Apochromat, M27 with DIC III Prism) objective (Zeiss). For both datasets, the spinning-disk confocal microscope used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 (Intelligent Imaging Innovations, Inc.). Images were acquired using a Photometrics Evolve, a back-illuminated EMCCD camera (512 × 512 pixels).

The spinning-disk confocal microscope (spinning-disk confocal) used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 (Intelligent Imaging Innovations, Inc.). Images were acquired using either an Orca Flash 4 sCMOS camera (chip size 2,048 × 2,048; Hamamatsu Photonics) or an Evolve 512 EMCCD camera (chip size 512 × 512; Photometrics). Objectives used were a 40x water (NA 1.1, LD C-Apochromat, Zeiss), a 63 × oil (NA 1.4, Plan-Apochromat, M27 with DIC III Prism, Zeiss) and a 100x oil (NA 1.4 oil, Plan-Apochromat, M27) objective.
The structured illumination microscope (SIM) used was DeltaVision OMX v4 (GE Healthcare Life Sciences) fitted with a 60x Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases x three rotations). Emitted light was collected on a front-illuminated pco.edge sCMOS (pixel size 6.5 mm, readout speed 95 MHz; PCO AG) controlled by SoftWorx.
The confocal microscope used was a laser scanning confocal microscope LSM880 (Zeiss) equipped with an Airyscan detector (Carl Zeiss) and a 40x oil (NA 1.4) objective. The microscope was controlled using Zen Black (2.3), and the Airyscan was used in standard super-resolution mode.