MJG2401_LB

Time-lapse microscopy of MJG2401 grown on LB media at 37°C.Strains of interest were grown overnight in 5 mL of LB liquid broth shaking at 37°C to obtain a saturated culture. Cultures were back diluted 1,000-fold and grown to an early exponential phase (approximately 3 hours). Cells were concentrated by centrifugation, when necessary, at 5,000 <i>g </i>for 1 minute, before being resuspended in 100 μL of media. Cells were immobilized on agarose pads by spotting 0.5 μL of concentrated culture on the pad, then inverting the pad onto a glass bottom petri dish for imaging. Agarose pads for microscopy were constructed out of LB or MOPS minimal medium containing 1.5% agarose (Thermo Fisher cat. #16500500). Imaging was performed using equipment available at the University of Alabama at Birmingham High-Resolution Imaging Facility (HRIF); a Nikon Ti2 inverted fluorescencemicroscope with a tandem galvano and Nikon A1R-HD25 resonance scanner up to 30 1,024 × 1,024 images/s.Tokai Hit incubation stage chamber was used to heat samples and objectives to 37°C to facilitate the growth of bacteria for live imaging. Images were captured at 60× or 100× magnificationin both transmitted light differentialinterference contrast image and GFP or appropriate fluorescentchannels as needed. Automated time-lapse imaging was performed at 37°C, and motorized x, y, and z tracking was controlled and automated by acquisition software Nis Elements 5.0 Imaging Software available in the HRIF at UAB.

本数据集记录了于37℃ LB培养基中培养的MJG2401菌株的延时显微成像结果。目标菌株先于5 mL LB液体培养基中，在37℃摇床培养过夜以获得饱和培养液；随后将培养液以1000倍比例稀释，继续培养至早期指数生长期（约3小时）。必要时，将菌体以5000×g离心1分钟进行浓缩，并重悬于100 μL培养基中。取0.5 μL浓缩后的菌液滴加至琼脂糖胶垫上，随后将胶垫倒置固定于玻璃底培养皿中以进行成像。显微成像所用的琼脂糖胶垫由含1.5%琼脂糖的LB培养基或MOPS基本培养基配制而成（赛默飞世尔科技（Thermo Fisher），货号：16500500）。成像实验于阿拉巴马大学伯明翰分校高分辨成像中心（High-Resolution Imaging Facility, HRIF）开展，所用设备为尼康（Nikon）Ti2倒置荧光显微镜，搭配串联检流计模块与尼康A1R-HD25共振扫描仪，成像帧率最高可达30帧/秒（单帧分辨率为1024×1024像素）。使用托凯希特（Tokai Hit）孵育载物台腔室将样品与物镜加热至37℃，以维持细菌生长状态，实现活细胞成像。图像采集采用60×或100×放大倍率，分别获取透射光微分干涉差（DIC, Differential Interference Contrast）图像，以及根据需求采集绿色荧光蛋白（GFP, Green Fluorescent Protein）或其他适配荧光通道的图像。本次实验采用37℃下的自动化延时成像模式，电动X/Y/Z轴追踪功能由阿拉巴马大学伯明翰分校高分辨成像中心配备的NIS-Elements 5.0成像采集软件控制并实现自动化。