This workshop aims to show the participants how to perform 3D Single Molecule Localization Microscopy on reconstituted systems: purified proteins and model membranes having shapes like those observed in endocytosis Endocytosis is essential for numerous cellular functions; when failing, it causes many human diseases including cancers and neurodegenerative diseases. Mechanistically, endocytosis begins with deforming a flat donor membrane into a thin tubule or a vesicular bud; then membrane fission occurs to separate the nascent carrier from the donor membrane. The typical diameter of the endocytic neck is around 100 nm with various neck lengths. Thus, the endocytic necks have a saddle-like shape. Notably, several membrane curvature sensor BAR proteins have been implicated in endocytosis, for instance, by self-assembling at the neck to constrict the tubular neck and by regulating actin assembly at the neck to facilitate neck fission. In our project, we aim to reveal the molecular organization of BAR proteins at saddle-shaped membranes as in endocytic necks. We have reconstituted in vitro systems composed of purified BAR proteins and model lipid bilayers having endocytic-neck shapes. Given the nanometer-scale membrane necks, to reveal the molecular organization of BAR proteins at the neck, it is absolutely required to use 3D Single Molecule Localization Microscopy (SMLM). In this workshop, we will demonstrate the following work packages (1) SMLM detection of membrane shapes using membrane reporter Cy5-PC and DiI (2) SMLM detection of AX647 labelled BAR proteins at saddle-shaped membranes (3) Single particle tracking of lipids on membranes using Atto647N-PE to reveal lipid dynamics