Cfi apo 100

Fluorescent proteins were visualized with a CSU-X1 spinning disk confocal on a Nikon Eclipse Ti inverted microscope with an Andora Clara digital camera using CFI APO 100× oil TIRF objective. Images were collected using differential interference contrast (DIC), 488-nm excitation (GFP), and 561-nm excitation (RFP) and analyzed with NIS-Elements software 4.10 (Nikon) and Fiji [87 (link)].

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.