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

成像是一种强大的工具，用于理解和利用细胞和组织内部的基本过程。自从显微镜在16世纪后期被发现以来，它在放大倍率和灵敏度方面取得了许多技术进步。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个分子。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。如果使用共聚焦显微镜，则来自相邻切片的最小背景光使最终图像的分辨率和灵敏度精确。这些进步有助于阐明分子之间的相互作用（例如抗菌剂与其靶标），宿主细胞与入侵微生物之间的相互作用，肿瘤内的过程以及细菌/真菌的工程，以产生对我们有用的产品（例如生物燃料）。目前，在筛选(i)细胞以确定我们可以利用的细胞，或（ii）我们可以开发成有效药物的新化合物方面存在瓶颈。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。开发人员现在正在构建高分辨率显微镜，可以自动处理多个样品，从而实现高通量筛选。我们请求支持购买最新一代显微镜之一：高通量，高含量成像系统（HCS）。最近，HCS的逐步改进为市场带来了由机器学习指导的具有共聚焦和超分辨率功能的HCS。人工智能（AI）驱动的图像采集的强大之处在于，HCS可以以低分辨率扫描多个样本，并经过训练，专注于高分辨率成像的有趣区域。这样，提高了筛选的速度，自动化减少了误差。这种新一代显微镜的两个附加功能与我们打算进行的研究特别相关。首先，共聚焦HCS具有可灭菌的样品夹。我们将利用这一点，将HCS安装在具有研究传染性微生物所需的安全容器的实验室中。其次，设备包括一个机柜，我们可以控制环境。这将使我们能够提供维持所研究系统的最佳条件，例如低氧或高氧/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第一个这样的设施。我们的申请得到了米德兰兹大学创新网络以及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，因为与它合作的31所大学和60多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将对新药（抗生素、抗病毒药物、抗生物膜药物、杀菌剂、抗肿瘤药物）和利用微生物生产的可开发产品产生真正的影响。这些进步将改善国民的健康和财富。HCS将由一个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一系列显微镜方面具有良好的记录。SLIM将扩大其全面的培训计划，以确保用户完全熟练地处理HCS，从而支持他们的职业发展，并最大限度地发挥HCS输出的潜力。HCS的可用性将通过设备目录和网页向研究界和工业界公布。制作的图像将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.