N storm module

Fixed U2OS cells with tubulin immunolabelled by Alexa 647 were imaged by Nikon Eclipse Ti inverted epifluorescence microscope with NSTORM module (Nikon, Japan). The sample was illuminated in widefield mode by a 640 nm laser line through a 100× magnifying oil lens of 1.49 numerical aperture (HP Apo TIRF, Nikon, Japan). Optionally, simultaneous illumination by a 405 nm laser line was used to further promote the switching of the fluorophore between its bright and dark states. Stacks of 10,000 images were captured by Hamamatsu Orca-flash 4.0 camera at 100 frames per second rate. The total magnification of the microscope was adjusted by additional lenses to correspond to 108 nm per pixel. The cells were kindly provided by Dr. Ivan Novotný (Institute of Molecular Genetics of ASCR). They were kept in phosphate buffer which was replaced with STORM switching buffer⁷ prior to imaging. The signal to background ratio for this data is in the range 3 to 7.

Single-molecule imaging experiments were conducted on a custom-built Nikon Ti microscope. The microscope is equipped with a ×100 Oil-immersion objective lens (N.A. = 1.49), a multi-band dichroic (405/488/561/633 BrightLine quad-band bandpass filter, Semrock, USA) and a piezo z-stage (ASI, USA), a filter wheel (Sutter Instrument, USA) and a stage top incubator (Tokai Hit, Japan). The lasers were focused into the back pupil plane of the objective to generate wide-field illumination. A Nikon N-STORM module was used steer the incidence angle of the laser for generating inclined illumination. The emission was collected by the same objective passing through an emission filter (617/73, Semrock) in front of sCMOS camera (Prime 95B, Teledyne Photometrics). The microscope, lasers and the camera were controlled through NIS-Elements (Nikon, USA).