V700 flatbed scanner

In the microscopy Sandwich setup, seedlings were placed onto a thin layer of ½ MS medium placed inside a custom 3D printed chambered coverglass (24 × 50 mm). The seedlings were allowed to recover vertically for at least 30 min before gravistimulation. In the Through setup, the roots were growing unobstructed on the surface of the agar, and the imaging was performed through the coverglass and the agar.
Imaging was performed using a vertical stage (von Wangenheim et al., 2017 (link)) Zeiss Axio Observer 7 coupled to a Yokogawa CSU-W1-T2 spinning disk unit with 50 μm pinholes and equipped with a VS-HOM1000 excitation light homogenizer (Visitron Systems). Images were acquired using the VisiView software (Visitron Systems) and Zen Blue (Zeiss). We used the Zeiss Plan-Apochromat 20 × /0.8, Plan-Apochromat 10 × /0.45 and EC Plan-Neofluar 5 × /0.16 objectives. Brightfield signal was retrieved using a PRIME-95B Back-Illuminated sCMOS Camera (1200 × 1200 px; Photometrics), Orca Flash 4.0 V3 (2048 × 2048 px; Hamamatsu) and the camera from a LSM 700 confocal microscope (Zeiss).
For the scanner setup, seedlings were transferred to ½ MS, 1% sucrose (unless specified otherwise) and imaged every 30 min using an Epson Perfection v370, v600, or v700 flatbed scanner. The procedure followed to automate the scanning process is described in the Supplementary User Manual.