Live 3D Confocal Imaging in real time with high throughput, multipoint, targeted acquisition and AI-assisted quantification

成像是一种用于理解和利用细胞和组织内基本过程的强大工具。自从显微镜在15世纪末被发现以来，它在放大和灵敏度方面已经有了许多技术进步。现在，使用超分辨率显微镜可以实时跟踪小到细菌的细胞内的单分子。还可以拍摄一系列垂直图像(光学切片)，以建立组织样本和微生物的精确3D重建。如果这是使用共聚焦显微镜进行的，则来自相邻切片的背景光最少，从而使最终图像的分辨率和灵敏度精确。这些进展有助于阐明分子之间的相互作用(例如，抗菌剂与其靶标之间的相互作用)，宿主细胞与入侵微生物之间的相互作用，肿瘤内的过程，以及制造对我们有用的产品的细菌/真菌工程(例如生物燃料)。目前，在筛选(I)细胞以确定我们可以开发哪些细胞，或者(Ii)我们可以开发成有效药物的新化合物方面存在一个瓶颈。造成延迟的原因是最高分辨率的显微镜一次只能观察一个样品。开发人员现在正在建造能够自动处理多个样本的高分辨率显微镜，从而实现高通量筛选。我们正在请求支持，以购买最新一代的显微镜：高通量、高内容成像系统(HCS)。最近，HCS的阶跃变化改进为市场带来了由机器学习指导的具有共焦和超分辨率能力的HCS。人工智能(AI)驱动的图像采集的力量在于，HCS可以低分辨率扫描多个样本，并被训练成专注于感兴趣的区域进行高分辨率成像。这样，筛选的速度加快了，自动化减少了错误。这一新一代显微镜的另外两个特点与我们建议进行的研究特别相关。首先，共焦HCS具有可消毒的样品固定器。我们将通过在实验室安装HCS来利用这一点，该实验室具有研究传染性微生物所需的安全容器。其次，设备包括一个我们可以控制环境的柜子。这将使我们能够为维护研究中的系统提供最佳条件，例如低或高氧气/湿度/最佳温度(例如不同的微生物和3D组织模型)，并允许我们通过进行高分辨率的延时成像来跟踪细胞过程。所要求的高规格设备将是中部地区的第一个这样的设施。我们的申请得到了米德兰大学创新网络的支持，也得到了行业合作伙伴的大力支持。值得注意的是，国家生物膜创新中心正在支持我们的申请，因为它合作的31所大学和60家公司将能够在他们的生物膜研究中利用HCS。我们预测，共聚焦HCS将对利用微生物产生的新药(抗生素、抗病毒、抗生物膜、杀菌剂、抗肿瘤)和可开发产品的流水线产生真正的影响。这些进步将改善国家的健康和财富。中心将由一支经验丰富的成像团队(SLIM)管理，该团队在维护和支持内部和外部科学家使用显微镜组合方面有着良好的记录。SLIM将扩大其全面的培训计划，以确保用户在处理HCS方面拥有充分的技能，从而支持他们的职业发展，并最大限度地发挥HCS产出的潜力。将通过设备目录和网页向研究界和产业界宣传HCS的供应情况。所创建的图像将被纳入正在进行的外联活动。

Imaging is a powerful tool used to understand and exploit the fundamental processes within cells and tissues. Since the microscope was discovered in the late 1500s, there have been many technical advances in the magnification and sensitivity it can achieve. It is now possible to track single molecules in real time within cells as small as bacteria using super resolution microscopy. It is also possible to take a series of vertical images (optical slices) to build up a precise 3D reconstruction of tissue samples and microbes. If this is performed using confocal microscopy there is minimal background light from adjacent slices making the resolution and sensitivity of the final images precise. These advances are facilitating the elucidation of interactions between molecules (e.g. an antimicrobial with its target), interactions between host cells and invading microbes, processes within tumours and also the engineering of bacteria/fungi to generate products of use to us (e.g. biofuels). Currently there is a bottleneck in the screening of (i) cells to identify which we can exploit, or (ii) novel compounds that we could develop into effective drugs. The delay is caused because the highest resolution microscopes only view one sample at a time. Developers are now building high resolution microscopes that process multiple samples automatically, enabling high throughput screening. We are requesting support to purchase one of the newest generation of microscopes: a high throughput, high content imaging system (HCS). Recently step change improvements in HCS have brought to the market HCS with confocal and super resolution capabilities that are guided by machine learning. The power of artificial intelligence (AI)-driven image acquisition is that the HCS can scan multiple samples at low resolution and be trained to focus in on interesting areas for high resolution imaging. In this way, the speed of the screening is increased and the automation reduces error.Two additional features of this new generation of microscopes are particularly relevant for the research we propose to undertake. Firstly, the confocal HCS has a sterilizable sample holder. We will exploit this by installing the HCS in a laboratory with the safety containment required for the study of infectious microbes. Secondly, the equipment includes a cabinet in which we can control the environment. This will enable us to provide the best conditions for maintaining the system under study e.g. low or high oxygen/humidity/optimal temperature (e.g. different microbes and 3D tissue models) and allow us to follow cellular process by undertaking time-lapse imaging at high resolution. Equipment with the high specification requested will be the first such facility in the Midlands. Our application has the support of the Midlands Innovation network of Universities as well as considerable support from industry partners. Notably, the National Biofilm Innovation Centre is supporting our application because the 31 universities and >60 companies that it partners with would be able to exploit the HCS in their biofilm research. We predict that a confocal HCS will make a real difference to the pipeline of new medicines (antibiotic, anti-viral, anti-biofilm, fungicides, anti-tumour) and exploitable products generated using microbes. These advances will improve the health and wealth of the nation. The HCS will be managed by an experienced imaging team (SLIM) with a track record in maintaining and supporting the use of a portfolio of microscopes by internal and external scientists. SLIM will expand its thorough training programme to ensure users are fully skilled in HCS handling, and thereby support their career development and maximise the potential of the output from the HCS. The availability of the HCS will be publicised through equipment catalogues and web pages to the research community and industry. The images created will be integrated into ongoing outreach activities.