Zt647rdc

Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, Oil). A 561 nm and 640 nm (MPB Communications Inc, 2W, DPSS-system) laser were used for excitation. The laser beam was passed through clean-up filters (Chroma Technology, ZET561/10, ZET642/20x) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc, ZT647rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET575lp, ET705/72m and ET665lp) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2 × 2 binning).

Fluorescence imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) with the Perfect Focus System, applying an objective-type total internal reflection fluorescence (TIRF) configuration with an oil-immersion objective (Apo SR TIRF 100×, NA 1.49, Oil). Two lasers were used for excitation: 561 nm (200 mW, Coherent Sapphire) or 640 nm (150 mW, Toptica iBeam smart). The laser beam was passed through cleanup filters (ZET561/10 or ZET642/20, Chroma Technology) and coupled into the microscope objective using a beam splitter (ZT561rdc or ZT647rdc, Chroma Technology). Fluorescence light was spectrally filtered with an emission filter (ET600/50m and ET575lp or ET705/72m and ET665lp, Chroma Technology) and imaged on an electron-multiplying charge-coupled device (EMCCD) camera (Andor iXon Ultra 897, used for Figs. 1a and 3c) or sCMOS camera (Andor Zyla 4.2, used for Figs. 3b, d, and 4) without further magnification, resulting in an effective pixel size of 160 nm (EMCCD) or 130 nm (sCMOS after 2 × 2 binning).