Confocal image acquisition system with capacity for robotic fluid additions: flexible tool for high-content screening

具有机器人液体添加能力的共焦图像采集系统：用于高内涵筛选的灵活工具

• 批准号: MR/X013383/1
• 负责人: Paola Vergani
• 金额: $ 48.37万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FX013383%2F1
• 关键词: Confocal; image; acquisition; system; capacity

Experiments investigating cell physiology and drug responses often exploit light-emitting (fluorescent) proteins and probes. Fluorescent probes, engineered to report on different cellular parameters, can be used as "biosensors". In the past, these experiments were conducted examining a small visual field under a microscope, manually adding compounds to dishes carrying cells, and selecting a small number of cells on which to measure changes. New image-acquisition systems allow this process to be done in an automated way. Cells are grown on plates with tens or hundreds of separate wells. At specified time points in the experiment, robotic arms add different compounds to the different wells, while images are captured by a camera which detects light of different colours, emitted by different biosensors. Small, localised changes in response to perturbations are captured rapidly and continuously over a period of time. Automated computer-based analysis of the images greatly increases the efficiency of work.Such image-acquisition systems have made it possible to increase the number of cells studied by orders of magnitude. This has eliminated experimenter bias and allowed us to focus on subsets of cells. Optical resolution has improved, so we can even study sub-cellular structures, called organelles. In addition, the possibility of using multiple biosensors simultaneously has allowed measurement of multiple characteristics in the same cell/organelle. Assays with multiple readouts are described as having "high-content". Furthermore many different conditions can be rapidly compared on a single plate, (e.g. composition of the fluid added, genetic makeup of cells, etc.). This allows rapid "screening" of a large number of compounds, for instance, which can be useful when developing drugs.The image-acquisition system we propose to buy will be used by many research groups, and will be available across the UCL campus. It will allow us to develop and run different high-content assays.One proposed study will explore potential new therapies for cancer. All our cells have mitochondria, organelles that burn fuels and generate packages of energy, readily available for the cell's needs. Mitochondria have their own genetic material, mtDNA, distinct from that in the cell's nucleus. Scientists have discovered that tumour cells very often have changes, called mutations, in their mtDNA, not present in the surrounding healthy tissues. These mtDNA mutations can be used as targeting labels, directing specialized enzymes (called mitoTALENs) to make damaging cuts in mtDNA from tumour cells, without affecting the nearby tissues. Cells with damaged mtDNA grow more slowly, and are more susceptible to chemotherapy drugs. Using high-content screening techniques, tumour and healthy cells will be treated with mitoTALENs under a variety of different conditions. The information gained will validate the approach, and lay foundations for future therapy development.Another lab will work on improving treatment for people with cystic fibrosis (CF). In CF the CFTR protein is missing or defective. CFTR regulates flow of anions (negatively charged chloride and bicarbonate ions) into and out of cells that line ducts of our body (airways, intestine, pancreas, liver etc). The flow of bicarbonate is especially important for controlling mucous secretions produced by these duct cells. CFTR-targeted drugs can help CF patients, but we know that, at least in liver ducts, current drugs restore chloride but not bicarbonate flow. We will generate a model anion flux biosensor system. This will allow us to rapidly monitor chloride and bicarbonate flow and to determine how CFTR drugs affect it for 62 different variants of CFTR found in patients. What we will learn about processes at the root of CF disease will help clinicians choose the best drugs for individual patients, and guide future drug development.

研究细胞生理学和药物反应的实验经常利用发光（荧光）蛋白质和探针。荧光探针被设计用来报告不同的细胞参数，可以用作“生物传感器”。过去，这些实验是在显微镜下检查小视野，手动将化合物添加到携带细胞的培养皿中，并选择少量细胞来测量变化。新的图像采集系统允许以自动化方式完成此过程。细胞在具有数十或数百个独立孔的平板上生长。在实验的指定时间点，机械臂向不同的孔中添加不同的化合物，同时摄像头捕捉图像，摄像头检测不同生物传感器发出的不同颜色的光。在一段时间内快速、连续地捕获响应扰动的微小局部变化。基于计算机的自动图像分析极大地提高了工作效率。此类图像采集系统使得研究的细胞数量增加几个数量级成为可能。这消除了实验者的偏见，使我们能够专注于细胞子集。光学分辨率得到了提高，因此我们甚至可以研究亚细胞结构，称为细胞器。此外，同时使用多个生物传感器的可能性使得可以测量同一细胞/细胞器中的多个特征。具有多个读数的测定被描述为具有“高含量”。此外，可以在单个板上快速比较许多不同的条件（例如添加的液体的成分、细胞的基因组成等）。例如，这可以快速“筛选”大量化合物，这在开发药物时非常有用。我们建议购买的图像采集系统将被许多研究小组使用，并将在整个伦敦大学学院校园内使用。它将允许我们开发和运行不同的高内涵检测。一项拟议的研究将探索潜在的癌症新疗法。我们所有的细胞都有线粒体，它们是燃烧燃料并产生能量包的细胞器，可随时满足细胞的需求。线粒体有自己的遗传物质，即线粒体DNA，与细胞核中的遗传物质不同。科学家发现，肿瘤细胞的线粒体 DNA 经常发生变化，称为突变，而周围健康组织中不存在这些变化。这些 mtDNA 突变可用作靶向标签，指导专门的酶（称为 mitoTALEN）对肿瘤细胞的 mtDNA 进行破坏性切割，而不影响附近的组织。线粒体 DNA 受损的细胞生长速度更慢，并且更容易受到化疗药物的影响。使用高内涵筛选技术，将在各种不同条件下用 mitoTALEN 处理肿瘤和健康细胞。获得的信息将验证该方法，并为未来的治疗开发奠定基础。另一个实验室将致力于改善囊性纤维化 (CF) 患者的治疗。 CF 中 CFTR 蛋白缺失或有缺陷。 CFTR 调节阴离子（带负电荷的氯离子和碳酸氢根离子）流入和流出人体管道（气道、肠道、胰腺、肝脏等）的细胞。碳酸氢盐的流动对于控制这些导管细胞产生的粘液分泌物尤其重要。 CFTR 靶向药物可以帮助 CF 患者，但我们知道，至少在肝管中，目前的药物可以恢复氯化物流动，但不能恢复碳酸氢盐流动。我们将生成一个模型阴离子通量生物传感器系统。这将使我们能够快速监测氯化物和碳酸氢盐的流量，并确定 CFTR 药物如何影响患者体内发现的 62 种不同的 CFTR 变体。我们将了解 CF 疾病根源的过程，这将帮助临床医生为个体患者选择最好的药物，并指导未来的药物开发。