Ff01 525 45

We spread Escherichia coli JM109 (DE3) cells transformed with the DNAs of mutant Kohinoor library on 95-mm LB agar plates and incubated them at 37°C for 16 h. We screened mutants with a high brightness and fast photoswitching by using an in-house-built illumination system [7 (link)]. We irradiated the Kohinoor mutants expressed in the E. coli cells with an excitation light at 475 nm from an LED light source (SPECTRA X Light Engine; Lumencor, Beaverton, OR, USA) and took the fluorescence through a bandpass filter (center wavelength, 525 nm; FF01-525/45; Semrock, Rochester, NY, USA) with a charge-coupled device (CCD) camera (01-QIClick-F-M12; QImaging, Surrey, BC, Canada). We measured the photoswitching with irradiation at 475 and 386 nm for on-switching and off-switching, respectively. We analyzed the data by ImageJ-Fiji software [20 (link)].

All SRM data were obtained with a custom-built single molecule imaging system. In brief, lasers emitting at wavelengths of 488 nm, 561 nm, and 647 nm were combined and introduced into the back of a Nikon Ti-U microscope. An f = 400 mm lens was used to focus the collimated laser light to the back aperture of a 60X TIRF objective to achieve total internal reflection illumination of the sample. Images were acquired with strict TIRF illumination to probe membrane proteins appropriately. During data acquisition a custom-built focus stabilization system based on the detection of the reflected excitation laser was used. A multi-edge polychroic mirror and emission filters FF01-525/45, FF01-605/64, and FF01-708/75 (Semrock, Rochester, NY, USA) were used to reflect laser light into the objective and clean up fluorescent signals. Images were collected using an iXon Ultra electron-multiplied charge coupled device (Andor, Abingdon, UK) with an EM gain of 200 and 30–40 ms exposure time. The power density of the 647 nm laser for DNA-PAINT(ERS) imaging was typically ~500 W cm⁻².