Uoplan water immersion objective

FCS served to measure the average radius of vesicle ensembles and the leakage of CF from lipid vesicles after subjection to an hypoosmotic gradient. In brief, the average residence time τ_D of Atto633-PPE labeled vesicles and the appearance of free CF visible as a second component in the autocorrelation function G(τ) of the fluorescence temporal signals from the confocal volume, was acquired using a commercial laser scanning microscope equipped with avalanche diodes (LSM 510 META Confocor 3 with a 40x-UOPLAN water immersion objective; Carl Zeiss). The confocal volume was calibrated using the residence time of rhodamine 6G in solution and its diffusion coefficient of 426 μm² s⁻¹,^{58 (link)} as previously described.^{59,60 (link)} To this end, we applied the standard model for one- or two-component free 3D diffusion.^{51,61 (link)}D was determined as ω²/4τ_D.

FCS was used to determine the channel density within the GUV membrane as previously described in Ref. ^{33 (link)}. In brief, the number of YFP-labeled AQP1 was derived from the autocorrelation function ( $G\left(\tau \right)$ ) of the temporal fluorescence intensity signal, detected with a commercial laser scanning microscope (LSM 510 META ConfoCor 3 with a 40x-UOPLAN water immersion objective; Carl Zeiss, Jena, Germany) using avalanche photodiodes.