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

研究细胞生理和药物反应的实验经常使用发光(荧光)蛋白质和探针。荧光探针可以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，在携带细胞的培养皿中手动添加化合物，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的方式完成这一过程。细胞生长在有数十个或数百个分开的井的平板上。在实验中的特定时间点，机械臂向不同的井中添加不同的化合物，同时图像由相机捕捉，该相机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、连续地捕捉到响应扰动的小的、局部的变化。基于计算机的自动图像分析极大地提高了工作效率。这样的图像采集系统使研究细胞的数量以数量级增加成为可能。这消除了实验者的偏见，使我们能够专注于细胞的子集。光学分辨率提高了，所以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允许在同一细胞/细胞器中测量多种特征。具有多个读数的分析被描述为具有“高含量”。此外，许多不同的条件可以在一个平板上进行快速比较(例如，添加的液体的组成、细胞的遗传组成等)。例如，这可以对大量化合物进行快速“筛选”，这在开发药物时可能很有用。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。这将使我们能够开发和运行不同的高含量分析。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，即燃烧燃料和产生能量包的细胞器，随时可以满足细胞的需要。线粒体有自己的遗传物质mtDNA，与细胞核中的遗传物质不同。科学家们发现，肿瘤细胞的线粒体DNA经常会发生变化，称为突变，而周围的健康组织中没有这种变化。这些mtDNA突变可以用作靶向标记，指导特定的酶(称为mitoTALEN)对肿瘤细胞的mtDNA进行破坏性切割，而不会影响附近的组织。线粒体DNA受损的细胞生长更慢，对化疗药物更敏感。利用高含量的筛选技术，肿瘤和健康细胞将在各种不同的条件下用mitoTALEans进行治疗。所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患者的治疗。在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(呼吸道、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特别重要。CFTR靶向药物可以帮助CF患者，但我们知道，至少在肝管内，目前的药物可以恢复氯化物，但不能恢复碳酸氢盐的流动。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动，并确定CFTR药物如何影响在患者中发现的62种不同的CFTR变种。我们将了解的CF病的根本过程将帮助临床医生为个别患者选择最好的药物，并指导未来的药物开发。

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.