Lf405 488 561 635 a 000

A schematic of the optical setup used is presented in Fig. 1a. We used a Nikon Eclipse Ti inverted microscope with a Nikon Perfect Focus System. The excitation was performed thanks to five different lasers: 637 nm (Obis 637LX, 140 mW, Coherent), 561 nm (Genesis MX 561 STM, 500 mW), 532 nm (Verdi G5, 5 W, Coherent), 488 nm (Genesis MX 488 STM, 500 mW, Coherent), and 405 nm (Obis 405LX, 100 mW, Coherent). The corresponding 390/482/532/640 or 390/482/561/640 multiband filters (LF405/488/532/635-A-000 and LF405/488/561/635-A-000, Semrock) were used. The fluorescence was collected through a Nikon APO TIRF ×100 1.49 NA oil immersion objective lens, sent in the DAISY module and recorded on two halves of a 512 × 512-pixel EMCCD camera (iXon3, Andor). The camera was placed at the focal plane of the module of magnification 1.67 and the optical pixel size was ~100 nm. Finally, the imaging paths were calibrated in intensity to compensate the non-ideality of the 50–50 beam splitter, as well as the reflection on the cylindrical lens surface (this measurement was performed for each fluorescence wavelength). The object focal plane of the EPI path was typically at the coverslip (z = 0 nm) and the UAF path had two focal lines, at z = 0 nm and z = 800 nm for the y and x axes, respectively.

Super-resolution dSTORM measurements were performed on a custom-made inverted microscope based on a Nikon Eclipse Ti-E frame (Nikon Instruments Europe BV, Amsterdam, The Netherlands). After being conditioned (through spatial filtering via fiber coupling and beam expansion), the applied laser beams were focused onto the back focal plane of the microscope objective (Nikon CFI Apo 100×, NA = 1.49), which produced a collimated beam on the sample. All dSTORM images were captured with a linearly polarized beam and EPI illumination at an excitation wavelength of 647 nm (MPB Communications Inc., Pointe-Claire, QC, Canada: 647 nm, P_max = 300 mW). The laser intensity was controlled via an acousto-optic tunable filter (AOTF). Images were captured by an Andor iXon3 897 EMCCD camera (Andor: Belfast, UK; 512 × 512 pixels with 16 μm pixel size). Frame stacks for dSTORM super-resolution imaging were typically captured at a reduced image size (crop mode). Excitation and emission wavelengths were spectrally separated with a fluorescence filter set (Semrock, Rochester, NY, USA; LF405/488/561/635-A-000) and an additional emission filter (Semrock, BLP01-647R-25) in the detector arm. During the measurements, the perfect focus system of the microscope was used to keep the sample in focus with a precision of <30 nm.