Tirf illuminator

Twisting measurements were performed on a Ti-E inverted microscope (Nikon, NY, USA) with a 100X objective (NA, 1.40). A 488-nm Sapphire optically pumped semiconductor laser (OPSL; Coherent, CA, USA) was coupled with a TIRF illuminator (Nikon) attached to the microscope stand. Images were acquired with a DU885 EMCCD camera (Andor, CT, USA), and synchronization between components was achieved using μManager (52 (link)).

An Eclipse Ti inverted microscope (Nikon) with a TIRF system and iXon electron-multiplying charged-coupled device camera (Andor Technology) was used for imaging. TIRF microscopy was performed with a Nikon 100 × 1.49 numerical aperture oil-immersion TIRF objective, a TIRF illuminator, a Perfect Focus system, a motorized stage, and a U-N4S four-laser unit as a laser source (Nikon). The laser unit has the solid-state lasers for the 488 nm, 561 nm, and 640 nm channels and was controlled using a built-in acousto-optic tunable filter. The laser powers of 488 nm laser, 561 nm laser, and 640 nm laser were set to 5.2, 6.9, and 7.8 mW respectively measured with the field aperture fully opened. The 405/488/561/638 nm Quad TIRF filter set (Chroma Technology Corp.), along with supplementary emission filters of 525/50 m, 600/50 m, 700/75 m for 488 nm, 561 nm, 640 nm channels, respectively, were used. Images are acquired using the Nikon NIS-Elements software with the exposure time of 50 ms.

The samples were illuminated using a 405-nm laser diode source (Coherent Cube) using an inverted microscope fitted with a TIRF illuminator (Nikon) and a × 100 oil immersion objective (NA 1.49, Nikon). The laser light was filtered using dichroic (Z405rdc Chroma) and emission filters (ET420LP Chroma). Single molecule fluorescence was collected using an EMCCD camera (Photometric Evolve 512). Each frame had a 100 ms exposure time, with ∼30 ms of dead time between acquisitions. SR maps were constructed from a minimum of 60,000 images and, on average, contain ∼4,000 successful localization points after suitable filtering. In order to account for sample drift scattered laser light from the sample was reflected by the dichroic mirror and collected via a second camera (QICam). A disk in each array of structures was used as a reference point and the scattered laser light was localized and used to correct the sample position.
During the measurement process, the illuminating laser was kept at low power (on the order of ∼10⁻¹ W cm⁻²) to ensure that in the presence of an enhanced EM field around our plasmonic structures, our dyes continued to operate in a linear response regime. The reader should note that this is one to several orders of magnitude less than conventional super-resolution microscopy techniques and as a result unenhanced molecules at the glass/sample interface are not observed.