Ronin single molecule

Overnight cultures were diluted 1:100 in LB supplemented with 0.1% L-Arabinose to overexpress Ronin, and grown until they reached an optical density (OD600) of 0.4-0.6. Cultures were induced with 1 mM IPTG for 30, 90 or 120 minutes before harvesting for microscopy. Cells were washed once with 1X Phosphate buffer saline (PBS) and resuspended in 700 µL of 1X PBS. Next, 30 µL of cell suspension were loaded onto a polylysine (Sigma P8920) coated coverslip and fixed in 4% paraformaldehyde for 10 minutes at room temperature. To minimize nonspecific staining, the sample was blocked with 5% BSA for 15 minutes at room temperature. To visualize the membranes, samples were stained with 10 µg/mL WGA AF647 in 1X PBS for 15 minutes, and washed three times with 1X PBS. ONI dSTORM imaging buffer was added prior to imaging.
Cells were imaged with an ONI NanoImager microscope (Oxford, UK). The microscope was equipped with 100x oil immersion objective with a numerical aperture of 1.45 and lasers with the following specifications: 405 nm/150 mW, 488 nm/1 W, 560 nm/500 mW, and 640 nm/1 W. ONI Bcube B dSTORM buffer was used as the imaging buffer. mNeonGreen was excited with a 488 nm laser at 112 mW, while WGA AF647 was excited using a 640 nm laser at 100 mW of power at the objective. Emission channels were separated using 640 dichroic and recorded as two separate images. Simultaneous excitation of dyes was performed with a 30 millisecond exposure time over 20,000 frames, employing HILO (Highly Inclined and Laminated Optical sheet) illumination technique. The acquisition was performed using NimOS 19.5 software, which incorporates real-time localization to generate super-resolution images. To enhance image quality, NimOS built-in drift correction was applied to single molecule images, and a localization precision filter between 0 and 20 nm was employed. Additionally, the first 100 frames were disregarded to ensure stability before analysis. For analysis of the localization distribution, the line histogram tool available in NimOS was utilized to extract the localization across the width of 30 randomly selected cells. The resulting localization distribution over distance data was exported as a .CSV file for further analysis using GraphPad Prism 10.