Images were acquired using total internal reflection fluorescence microscopy (TIRF) configuration on a Nikon Eclipse Ti microscope with the following available laser lines: 405, 488, 561, and 657 nm and Spectra EX (Lumencor). TIRF APO 100x 1.49 N.A. objective was used for acquiring images. Emission/excitation filters used were as follows: GFP (mirror: 498–537 nm and 565–624 nm; excitation: 450–490 nm and 545–555 nm; emission: peak 525 nm, range 30 nm) and mCherry (mirror: 430–470, 501–539, and 567–627 nm; excitation: 395–415, 475–495, and 540–560 nm; emission: peak 605 nm, range 15 nm) or Continuous STORM (mirror: 420–481, 497–553, 575–628, and 667–792 nm; excitation: 387–417, 483–494, 557–570, and 636–661 nm; emission: 422–478, 502–549, 581–625, and 674–786 nm).
A polarized evanescent wave excites the fluorophores. In the emission pathway, the emitted light was split into p and s polarization components with a polarization beam splitter (Laser Beamsplitter zt 561 sprdc), placed into an Optosplit III(Cairn Research). The orthogonal images (Ipa and Ipe) were projected onto two separate fields of view, manually aligned, and captured with a Teledyne Photometrics 95B 22 mm camera. The polarization of the evanescent field was verified by measuring the extinction coefficient.
A polarized evanescent wave excites the fluorophores. In the emission pathway, the emitted light was split into p and s polarization components with a polarization beam splitter (Laser Beamsplitter zt 561 sprdc), placed into an Optosplit III(Cairn Research). The orthogonal images (Ipa and Ipe) were projected onto two separate fields of view, manually aligned, and captured with a Teledyne Photometrics 95B 22 mm camera. The polarization of the evanescent field was verified by measuring the extinction coefficient.