Csu w1 t2

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.

Root surface pH was measured using the ratiometric Fluorescein-5-(and-6)-Sulfonic Acid, Trisodium Salt (FS) (Invitrogen™ F1130).⁸¹ (link) Five-day-old Arabidopsis seedlings were transferred to unbuffered ½ MS medium containing 50 μM FS dye and either 0 or 100 nM IAA. Seedlings were allowed to recover on a vertical spinning disk microscope for 20 minutes after transfer to the microscope chamber. Imaging was performed using a vertical stage Zeiss Axio Observer 7 microscope coupled to a Yokogawa CSU-W1-T2 spinning disk unit with 50 μm pinholes, equipped with a VS-HOM1000 excitation light homogenizer (Visitron Systems). Images were acquired using VisiView software (Visitron Systems, v.4.4.0.14). We used a Zeiss Plan-Apochromat ×10/0.45 objective. FS was excited by 405 and 488 nm laser. The 488/405 nm fluorescence emission ratio along the root was calculated using the ATR software.⁸¹ (link)