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）是一种被广泛认可且日益重要的用于鉴定调节生物过程的化合物的方法。由此鉴定的小分子化合物可以是用于解剖复杂生物学途径的强有力的分子探针，并且也可以是作为治疗剂开发的潜在先导。RNA干扰（RNAi）是探测基因功能的另一种强大工具，使用HTS技术，可以系统地实施以筛选整个基因组的特定分子功能或潜在的治疗靶点。ICCB-Longwood Screening Facility（ICCB-L）Research Core为哈佛医学院（HMS）、HMS附属医院、更广泛的波士顿地区研究社区成员以及全国其他实验室提供小分子和RNAi HTS资源。 在这里，我们请求资助购买一台Molecular Devices IsoCyte激光扫描细胞仪，用于ICCB-L的高通量筛选。虽然这种类型的仪器在美国其他地方的学术筛选中心使用，目前在HMS或邻近的研究医院中没有类似的仪器。如本申请中所述，IsoCyte将为NIH资助的大型社区带来新的和急需的测定功能。 IsoCyte生成微孔板威尔斯孔的低分辨率图像。使用具有大焦深的光学头和多个光电倍增管收集来自激光激发样品的荧光发射。光散射模式有助于生成类似于来自非荧光样品的明场图像的图像。图像分析算法在捕获图像时同时运行，以检测对象（细胞）并在多达4个发射通道中量化强度。IsoCyte在多模式酶标仪和筛选显微镜之间的检测读数中占据了一个有用的利基。IsoCyte相对于酶标仪的主要特点包括IsoCyte的灵敏度大大增强和收集单细胞数据的能力。IsoCyte在捕获和分析低分辨率图像方面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体（如斑马鱼）的理想选择。这样的屏幕是不可行的使用传统的筛选显微镜。该提案反映了IsoCyte的许多潜在应用，包括针对传染病，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。由于其位于ICCB-L研究中心，该仪器将得到良好的维护，良好的管理，并在新用户招募时高度可访问。

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.