High throughput optical mesoscopy for quantitative screening of novel antimicrobial compounds

抗生素耐药性是我们今天面临的最大健康挑战之一。迫切需要开展研究，以支持快速发现和筛选新型抗生素，并减少将这些抗生素推向市场所需的时间和成本。该项目旨在开发关键的基础成像和分析技术，以有效筛选和研究新的抗菌候选物，特别是抗菌肽（AMP），这可能是最有前途的一类抗菌剂。现有的测定法如最小抑菌浓度（MIC）的重现性差，并且不能提供关于AMP的作用模式和细菌应答的多样性的详细信息。目前NPL使用的高分辨率光学成像方法（如超分辨率显微镜）只能对一小部分细胞进行详细的可视化，从而导致低通量和测量统计数据不佳。该项目旨在利用高通量成像和相关的图像分析技术来解决这些限制，并为评估抗菌剂提供强大的新能力。麦康奈尔教授开发了一种新的巨型透镜，称为Mesolens，它可以在衍射极限分辨率的单一图像中显示数百或数千个哺乳动物细胞的图像。我们的目标是扩展Mesolens的能力，以支持使用全内反射荧光（TIRF）显微镜和结构照明显微镜（SIM）的超分辨率成像模式。这些方法将在横向方向上将空间分辨率提高约2倍，在轴向方向上将空间分辨率提高约4倍，以同时解析数千个细胞中AMP的作用模式（例如膜孔形成），并使AMP对模型膜的作用可视化。NPL将超分辨率介观与免费成像和生物物理测量相结合，将为AMP作用机制提供新的见解，并允许以前所未有的灵敏度和可靠性筛选候选化合物。

Antimicrobial Resistance is one the biggest health challenges facing us today. Research is urgently required to support the rapid discovery and screening of novel antibiotics and to reduce the time and cost involved in bringing these to market. This project seeks to develop key underpinning imaging and analysis techniques to allow effective screening and study of novel antimicrobial candidates, specifically antimicrobial peptides (AMPs) which represent possibly the most promising class of antimicrobial agents. Existing assays such as minimum inhibitory concentration (MIC) suffer from poor reproducibility and cannot provide detailed information about the mode of action of AMPs and the diversity of bacterial responses. Current high resolution optical imaging approaches used at NPL (such as super-resolution microscopy) allow detailed visualisation of only a small population of cells, resulting in low throughput and poor measurement statistics.This project seeks to leverage high throughput imaging and associated image analysis techniques to solve these limitations and provide a powerful new capability for assessing antimicrobials. Prof McConnell has developed a new giant lens called the Mesolens which gives images of hundreds or thousands of mammalian cells in a single image with diffraction-limited resolution. We aim here to extend the capability of the Mesolens to support super-resolution imaging modes using total internal reflection fluorescence (TIRF) microscopy and structured illumination microscopy (SIM). These methods will improve the spatial resolution by a factor of approximately 2 in the lateral direction and 4 in the axial direction to resolve the mode of action of AMPs (such as membrane pore formation) in thousands of cells simultaneously and enable visualisation of AMP action on model membranes. Combining super-resolution mesoscopy with complimentary imaging and biophysical measurements at NPL will provide new insights into the mechanisms of AMP action and allow screening of candidate compounds with unprecedented sensitivity and reliability.