Eclipe ti

Longitudinal hand sections of in vitro-cultivated bud-bearing stem segments of pAtPIN1::AtPIN1-GFP-expressing tomato line were observed using a confocal laser scanning microscope (NIKON Eclipe Ti) with a 20× water immersion objective (excitation wavelength 488nm, emission spectra between 500 and 550nm). Laser power remained unchanged throughout the experiment. Quantification of the GFP signal was performed on 2D images using ImageJ software. Representative micrographs are given in Supplementary Figure S4. Integrated density of grey was determined on the 10 most intensely polarized plasma membrane poles of cells for each sample. The results are the means of three to four for replicates for each condition.

B cells from Lifeact-GFP MD4 mice were settled onto a PAA gel coated with either BSA or HEL. Cells were allowed to settle for 10 min before imaging with an inverted spinning disk confocal microscope (Eclipe Ti Nikon/Roper Spinning head) equipped with a × 40 Water immersion objective 1.4 NA and a CoolSNAP HQ2 camera (pixel size 6.4  μm) with Metamorph software (Molecular Device, France). Time lapse were typically 1 image/6 s, taking 10 images stack with dz = 0.4 μm, and last 6 min. The acquisition were bleach-corrected and projected in z, before cropping the cells. Patches were tracked, excluding the ones on the cortex, using ImageJ (TrackMate). Tracks were further analyzed on Matlab to extract the diffusion coefficient D (on tracks of length n frames (n>3) it was obtained as a linear fit without offset of the first max(10, n) points of the mean square displacement), the duration, and the localization relative to the center of the synapse. Maps were obtained as done for the beads map (using a gaussian kernel).