Laser scanning cytometer for high throughput screening

描述(由申请人提供)：小分子文库的高通量筛选(HTS)是一种被广泛认可的、日益重要的鉴定调节生物过程的化合物的方法。由此确定的小分子化合物可以成为解剖复杂生物途径的强大分子探针，也可能成为潜在的治疗药物开发方向。RNA干扰(RNAi)是另一种探测基因功能的强大工具，利用HTS技术，可以系统地实现对整个基因组的特定分子功能或潜在治疗靶点的筛选。ICCB-Longwood筛查设施(ICCB-L)研究核心为哈佛医学院、附属医院、波士顿地区更广泛的研究社区成员和全国其他实验室提供小分子和RNAi高温超导资源。在此，我们申请资金购买分子设备公司的IsoCyte激光扫描细胞仪，用于ICCB-L的高通量筛查。虽然这种类型的仪器在美国其他地方的学术筛查中心使用，但HMS或邻近的研究医院目前还没有类似的仪器。如本申请中所述，IsoCyte将为美国国立卫生研究院资助的大型社区带来新的和急需的分析能力。IsoCyte生成微板井的低分辨率图像。使用大焦深的光学头和多个光电倍增管收集激光激发的样品的荧光发射。光散射模式便于从非荧光样本生成类似于明场图像的图像。图像分析算法在捕获图像时并行运行，以检测对象(细胞)并量化多达4个发射通道中的强度。IsoCyte在多模式平板读取器和筛选显微镜之间的检测读数方面占据了一个有用的利基市场。与平板读取器相比，IsoCyte的主要特点包括IsoCyte的灵敏度大大增强，以及收集单细胞数据的能力。在捕捉和分析低分辨率图像方面，IsoCyte比目前使用的筛选显微镜快5-10倍。由于其大景深，IsoCyte是自动成像整个生物(如斑马鱼)的理想设备。使用传统的筛查显微镜，这种筛查是不可行的。这项提案反映了IsoCyte的许多潜在应用，包括涉及传染病、神经生物学、癌症生物学和细胞死亡领域的广泛主题的具体项目。由于它位于ICCB-L研究核心，该仪器将得到良好的维护和管理，并在新用户招募时高度可用。

DESCRIPTION (provided by applicant): High-throughput screening (HTS) of small molecule libraries is a widely recognized and increasingly important method for identifying compounds that modulate biological processes. Small molecule compounds thus identified can be powerful molecular probes for dissecting complex biological pathways, and may also be potential leads for development as therapeutics. RNA interference (RNAi) is another powerful tool for probing gene function and, using HTS technologies, can be implemented systematically to screen entire genomes for specific molecular functions or potential therapeutic targets. The ICCB-Longwood Screening Facility (ICCB-L) Research Core provides small molecule and RNAi HTS resources for Harvard Medical School (HMS), HMS-affiliated hospitals, members of the broader Boston-area research community, and other labs nationwide. Here we request funding to purchase a Molecular Devices IsoCyte laser scanning cytometer for use in high-throughput screening at ICCB-L. Although this type of instrument is in use in academic screening centers at other sites in the U.S., no similar instrument currently exists at HMS or in the adjacent research hospitals. As described in this application, the IsoCyte will bring new and much- needed assay capabilities to a large NIH-funded community. The IsoCyte generates low-resolution images of microplate wells. Fluorescence emissions from laser-excited samples are collected using an optical head with a large depth of focus and multiple photomultiplier tubes. A light-scatter mode facilitates generation of images that resemble brightfield images from non-fluorescent samples. Image analysis algorithms run concurrently as images are captured to detect objects (cells) and quantify intensities in up to 4 emission channels. The IsoCyte occupies a useful niche for assay readout between multimode plate readers and screening microscopes. Key features of the IsoCyte relative to plate readers include the greatly enhanced sensitivity of the IsoCyte and the ability to collect single-cell data. The IsoCyte is 5-10 times faster at capturing and analyzing low resolution images than screening microscopes currently in use. Because of its large depth of field, the IsoCyte is ideal for automating imaging of whole organisms such as zebrafish. Such screens are not feasible using conventional screening microscopes. Reflecting the many potential applications of the IsoCyte, this proposal includes specific projects addressing a wide range of topics in the areas of infectious disease, neurobiology, cancer biology, and cell death. Because of its location in the ICCB-L Research Core, the instrument will be well maintained, well managed, and highly accessible to new users as they are recruited.