N storm 4.0 system

All SMLM imaging was performed using the commercial N-STORM 4.0 system (Nikon) operated in TIRF mode through a 100 × 1.49 NA oil-immersion lens. Cells were imaged using a 647 nm excitation laser (~1.125 kW/cm²) and fluorescence captured on a EMCCD camera iXon3 DU-897E (Andor Technology Ltd.) at 25 °C. A quad bandpass emission filter set (Chroma 89902-ET-405/488/561/647 nm) was used to filter the fluorescence collected from the sample. Images were collected at 50 ms and 20 ms integration time for IRIS and dSTORM imaging respectively, and a total of 100,000 frames were typically recorded for image reconstruction.

30 μL of 10-fold diluted particles suspension was introduced into a flow chamber created with 24x24 mm glass cover slip (RS, France) attached with a two face Scotch tape to the edges of a glass slide (25x75 mm, Corning). The sample was incubated for 15 min. at RT and next 100 μL of purified water were introduced into the flow chamber to flush away unattached NPs.
Images were acquired using a Nikon N-STORM 4.0 system conFigd for total internal reflection (TIR) fluorescence, using a Perfect Focus System imaging. Excitation under the TIR conditions allowed to avoid illumination of out of focus, improving signal to noise ratio. DiI fluorophore was excited by illuminating the sample with a 5% power of 561 nm (80 mW) laser built into the microscope. During acquisition the integration time was 300 ms. Fluorescence was collected by means of a Nikon x100, 1.4 NA oil immersion objective and passed through a quad-band-pass dichroic filter (97335 Nikon). Images were recorded onto a 256 x 256-pixel region (pixel size 160 nm) of a Hamamatsu ORCA Flash 4.0 CMOS camera. The images were analyzed using ImageJ software. Briefly, the intensity threshold was set to filter the NPs in each image, and next the fluorescence intensity per particle was measured (for minimum 800 NPs) and plotted in a histogram graph.