A microscale sorting cytometer for cell-based screens

描述（由申请人提供）：我们建议开发一种结合显微成像和自动分选的细胞仪。细胞仪解决了基于细胞的遗传筛选的两个步骤：观察表型和检索“突变体”。我们的目标是能够对细胞进行排序，其中排序参数是基于从显微镜中获得的丰富信息：形态学，细胞内荧光，动力学。我们的细胞仪本质上是一个“主动”的盖盖，它使用半导体技术创建的可逆可寻址陷阱阵列来保持单个细胞的位置，以观察和释放选定的细胞。陷阱使用光学镊子的电子模拟-称为介电电泳-暂时保持贴壁细胞的位置，直到它们附着在衬底上。附着后，细胞仪是一个被动芯片，适合传统的直立荧光显微镜。活细胞测定完成后，可以释放处于选定陷阱中的单个细胞。我们研究的具体目标是(1)创建越来越复杂的捕获数组，从验证功能的4x4数组开始，表征排序的20x20数组，并以用于库屏幕的100x100数组结束；(2)创建控制系统，以提供恒定的温度，pH值和氧张力芯片与自动显微镜相结合；(3)研究我们的细胞仪对细胞健康的影响，并进行表型筛选。

DESCRIPTION (provided by applicant): We propose the development of a cytometer that combines microscopic imaging with automated sorting. The cytometer addresses two steps in cell-based genetic screens: observation of phenotypes and retrieval of "mutants". Our goal is to be able to sort cells where the sort parameter is based upon the rich information available from microscopy: morphology, intracellular fluorescence, dynamics. Our cytometer is essentially an "active" coverslip that uses an array of reversible addressable traps created with semiconductor technology to hold individual cells in place for observation and release selected cells. The traps use an electrical analogue of optical tweezers--termed dielectrophoresis--to transiently hold the adherent cells in place until they attach to the substrate. After attachment, the cytometer is a passive chip that fits under a conventional upright fluorescence microscope. After live-cell assays have been performed, individual cells in selected traps can be released. The specific aims of our research are to (1) create trapping arrays of increasing complexity, starting with a 4x4 array to verify function, a 20x20 array to characterize sorting, and ending in a 100x100 array for use in an library screen; (2) create the control systems to provide constant temperature, pH, and oxygen tension on-chip coupled with automated microscopy; (3) investigate effects of our cytometer on cell health and perform a phenotype screen.