Polarisation camera dSTORM datasets of actin in fixed HeLa cells labeled with phalloidin-Alexa Fluor 488

Polarisation camera dSTORM dataset of the actin of fixed HeLa cells labeled with phalloidin-Alexa Fluor 488.

Image acquisition was performed as follows:

Imaging was performed on a widefield microscope equipped with a polarisation camera (CS505MUP, Thorlabs). The sample was excited using a 488 nm laser at quasi-TIRF, with a measured power density at the image plane of 5.04 kW/cm^2. A single long-pass dichroic (Di02-R488, Semrock) was used to seperate fluorescence from the excitation. The emission was filtered using a long-pass (BLP01-488R, Semrock) and a bandpass filter (FF01-582/64, Semrock) before detection. An exposure time of 30 ms was used.

Samples were prepared as follows:

Cell culture: HeLa TDS cells were cultured in DMEM (Gibco, Invitrogen) supplemented with 10 % Fetal Bovine Serum (FBS, Life Technologies), 1 % penicillin/streptomycin (Life Technologies), and 1 % glutamine (Life Technologies) at 37 °C + 5 % CO_2. Cells were periodically tested for mycoplasma contamination and passaged 3 times per week. Cells were plated at low density on high-precision glass coverslips (MatTek, P35G-0.170-14-C) 1 day prior to fixation for dSTORM experiments.

Labeling: Cells were simultaneously fixed and permeabilized in cytoskeleton buffer (CBS, 10 mM MES, 138 mM KCl, 3 mM MgCl_2, 2 mM EGTA, 4.5 % sucrose w/v, pH 7.4), + 4 % paraformaldehyde (PFA) and 0.2 % Triton for 6 minutes at 37 °C, and further fixed in CBS + 4 % PFA for 14 minutes at 37 °C. Post-fixation, cells were washed x3 in PBST (PBS supplemented with 0.1 % Tween) and permeabilized a second time in PBS + 0.5 % Triton for 5 minutes at room temperature. The samples were then washed 3 times in PBST and blocked for 30 minutes in 5 % BSA. Cells were washed x3 in PBST and then incubated with Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen, 1:50 in PBS) for 1 h in the dark, followed by x2 washes in PBS. Prior to dSTORM imaging, PBS was replaced with dSTORM imaging buffer (base buffer consisting of 0.56 M glucose, 50 mM Tris (pH 8.5), and 10 mM NaCl supplemented with 5 U/mL pyranose oxidase (Sigma, P4234), 10 mM cysteamine (Sigma, 30070), 40 µg/mL catalase (Sigma, C100) and 2 mM cyclooctatetraene (Sigma, 138924).