Lu n4 four laser unit

The fixed samples were imaged on an automated inverted Nikon Ti-2 wide-field microscope equipped with ×60 1.4NA oil immersion objective lens (Nikon), Spectra X LED light engine (Lumencor), and Orca 4.0 v2 scMOS camera (Hamamatsu). The live cell experiments were performed on a custom microscope built around Nikon Ti-E stand. The excitation was through HTIRF (Nikon) with an LU-n4 four laser unit (Nikon) with solid state lasers with wavelengths 405, 488, 561, and 640 nm. The main dichroic was a quad band dichroic mirror (Chroma, ET-405/488/561/640 nm laser quad band set for TIRF applications). The imaging was done through the ×100 1.49NA oil immersion objective (Nikon). To achieve simultaneous 3-color imaging, we used a TriCam light splitter into three separate EMCCD cameras (Andor iXon Ultra 897) with ultraflat 2 mm thick imaging splitting dichroic mirrors (T565LPXR-UF2, T640LPXR-UF2). A band pass emission filter was placed in front of each camera, respectively (ET525/50 m, ET595/50 m, and ET655lp). The microscope was also equipped with an automated XY-stage with extra fine lead-screw pitch of 0.635 mm and 10 nm linear encoder resolution and a Piezo-Z stage (Applied Scientific Instrumentation) for fast Z-acquisition. The whole microscope was under the control of Nikon Elements for automation.