MDCK E-cadherin-GFP cells were observed 3 days after seeding on glass coverslips, and ≈12–24 h after confluency, in DMEM–FCS supplemented with 10 mM Hepes, in a 37 °C observation chamber (POCmini-chamber-system, Tempcontrol 37–2 Digital PeCon), on an IX71 inverted microscope (Olympus) with a high numerical aperture objective (63× oil-immersion, NA = 1.25, PlanNeofluar, Zeiss). Two-photon photobleaching was performed with a femto-second laser tuned at 878 nm, pumped by a 10W CW 532 nm laser (Mira 900 and Verdi; Coherent). The position of the beam was controlled by VM500 galvanometric mirrors (GSI Lumonics) and the photobleaching duration by a shutter LS200 (NnmLaser), driven by MetaMorph. The measured excitation Point-Spread Function (PSF) is an ellipsoid with a 0.5-μm diameter in the focal plane and a 1.5-μm extension along the optical axis. Fluorescence recovery was spatially resolved under 1-photon excitation by fast 3D wide field videomicroscopy, using a DG-4 monochromator set at 480 nm (Sutter), a Coolsnap HQ CCD camera (Roper Scientific), and a PiFoc piezo-driven objective actuator (Physik Instrumente). The FRAP experiment was performed as follows. One point in a junction was photobleached for 200 ms with a 30 mW average power excitation intensity as measured at the objective back pupil. A stack of 14 images with a 0.3-μm vertical spacing were acquired before (CIL# 24066) and after (CIL# 24067) photobleaching. This is the FRAP time-lapse video, with a time interval of 27 s. All three movies are from supporting information for Proc Natl Acad Sci. (209) 106: 7010-7015.