Orca flash 4 v3 camera

HeLa cells were cultured and plated in phenol red-free growth medium (Gibco). Before imaging, the cells were washed with PBS and compound PFF-1 (500 nM) was added to the medium 10 min prior to imaging. An adaptive optics plug and play accessory MicAO 3DSR for SMLM (Imagine Optic, France) was used. This module was inserted between the microscope side port and the sCMOS, Hamamatsu Orca Flash 4 v3 camera. As a second step, a controllable and aberration-free astigmatism was introduced to the point spread function (PSF) of single molecules, which allowed us to perform 3D SMLM based on astigmatism by determining the z positions of the fluorescent molecules based on the ellipticity of their PSF^{41 (link)}. The laser excitation was at 561 nm with a laser excitation intensity of 0.25 kW cm⁻².

For quantification of SNAPcell fluorescence, surface labeling and surface internalization, cells were imaged following staining on an Olympus IX81 inverted fluorescence microscope using a 63x/1.35 oil objective (Olympus) and the corresponding fluorescence fulter set. Images were captured using an Orca Flash 4 V3 camera (Hamamatsu) and MetaMorph software (Molecular Devices). For all experiments, specified fluorescence (i.e. SNAPcell ligand, surface signal) was normalized to FLAG fluorescence intensity (total Kir2.1 protein) for each cell to control for variations in the level of Kir2.1 expression following transient transfection. Exposures were set to ensure that no pixels were saturated. The exposure times were constant for each fluorescence channel across condition. For each transfection (independent replicate) at least 20 cells from three to four fields were imaged. In all experiments, data was combined from at least three independent replicates, unless otherwise specified.