Galvo mirrors

Patterned Supported Lipid Bilayers (pSLB) were obtained as described above on PDMS membranes, and the ring-containing membranes were mounted under an upright epifluorescence microscope (Nikon Ni, with Hamamatsu Orca Flash 4.0, v2). Images of pSLBs, obtained with either 15 or 30 s plasma cleaning, were acquired with a 60x water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca R2 camera. A small linear region of the pSLB was frapped by repeatedly scanning and focusing 180 fs pulses generated by a fiber laser (FemtoPower, Fianium) with central wavelength at 1064 nm at 20 MHz. A set of galvo mirrors (Thorlabs) and a telescope before the port of the microscope allowed to position and move (oscillations at 400 Hz) the diffraction-limited spot at a desired place on the bilayer. Once bleached, fluorescence recovery was monitored for 5 min. Time-lapse imaging during the pSLB photobleaching and its recovery after photobleaching was done with a home-made software (Labview 2011). Recovery of the intensity of the bleached lines were plotted either for the full line, or by separating the line into a left and a right area and assess whether the recovery was symmetric (Supplementary Fig. 1).

K-space emission was imaged using a Montana Instruments closed-cycle liquid He crysotat with piezo-controlled 3D-moveable sample stage, cryo-optic low-working distance 100x 0.9NA objective, vacuum housing, radiation shield, and local objective heater. A wavelength-tunable ultrafast laser (Toptica Photonics FemtoFiber Pro) was used for 488 nm excitation with 80 MHz repetition rate, guided into the cryo-optic with electrically-controlled Thorlabs Galvo mirrors. The sample emission was filtered through a Semrock tunable edge pass (set to 490 nm long pass) filter and directed via a 4 F imaging system into a Princeton Instruments Acton spectrometer and either a 512 (k-space) × 512 (wavelength) pixel or 1024 (k-space) × 1024 (wavelength) pixel Pixis camera.