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.将过夜培养的菌液以1:100的比例稀释于添加了0.1% L-阿拉伯糖的LB培养基中，以过表达Ronin蛋白，培养至光密度（OD600）为0.4~0.6。随后用1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导培养30、90或120分钟，之后收集菌体用于显微镜成像。菌体经1×磷酸盐缓冲液（PBS）洗涤一次，并重悬于700 μL 1×PBS中。取30 μL菌悬液滴加至包被有多聚赖氨酸（Sigma P8920）的盖玻片上，于室温下用4%多聚甲醛固定10分钟。为减少非特异性染色，样本于室温下用5%牛血清白蛋白（BSA）封闭15分钟。为可视化细胞膜，样本用10 μg/mL小麦胚芽凝集素AF647（WGA AF647）在1×PBS中染色15分钟，随后用1×PBS洗涤三次。成像前加入ONI dSTORM成像缓冲液。 使用ONI纳米成像显微镜（英国牛津）对菌体进行成像。该显微镜配备数值孔径为1.45的100×油浸物镜，以及如下参数的激光器：405 nm/150 mW、488 nm/1 W、560 nm/500 mW及640 nm/1 W。成像缓冲液采用ONI Bcube B dSTORM缓冲液。mNeonGreen用功率为112 mW的488 nm激光器激发，而WGA AF647则通过物镜处功率为100 mW的640 nm激光器激发。发射光路经640 nm二向色镜分离，并记录为两幅独立图像。采用高倾斜层叠光学片（HILO）照明技术，以30毫秒的曝光时长采集20000帧图像，实现染料的同步激发。成像过程使用NimOS 19.5软件完成，该软件集成实时定位功能以生成超分辨率图像。为提升图像质量，对单分子图像应用NimOS内置的漂移校正，并采用0~20 nm的定位精度过滤条件。此外，分析前舍弃前100帧图像以确保系统稳定性。对于定位分布的分析，使用NimOS内置的线直方图工具，提取30个随机选取的菌体宽度方向上的定位信息。所得距离定位分布数据导出为逗号分隔值（CSV）文件，用于后续采用GraphPad Prism 10进行的分析。