Microscopy-based cell separation, selective propagation, and selective single cell sequencing

细胞分析或细胞术对生物学研究的许多方面都是至关重要的。自20世纪60年代以来，流式细胞术一直是占主导地位的细胞术技术，因为它简单，高通量，并且可以与荧光激活的细胞分选相结合来分离特定的细胞群。然而，流式细胞术也有一些局限性，包括(1)细胞样品上的剪切应力；(2)加工过程中显著的细胞损失；(3)分析非球形细胞、精细细胞和细胞团的困难；(4)无法评估亚细胞结构和形态。使用荧光显微镜可以解决许多这些限制，但是显微镜具有不能分离靶细胞的关键限制，这反过来又排除了许多后续分析，例如繁殖形成克隆群体和单细胞测序。目前的分离显微镜鉴定细胞的技术涉及使用显微操作器物理转移单个细胞，但这种方法困难，低通量，容易污染。该研究计划的长期目标是开发基于显微镜的细胞分离、选择性繁殖和选择性单细胞测序技术。利用我们最近在这一领域的工作，以及在宽视场显微镜和图像处理方面的进展，我们相信我们的工作最终将使显微镜技术与流式细胞术竞争，成为细胞术、细胞分离和单细胞分析的标准工具。这项发现基金将培养研究生、本科生和博士后水平的hqp。参与拟议研究项目的学员将在一个充满活力和多学科的公平、多样和包容的环境中接受世界一流的培训。具体的研究和培训领域包括光学、显微镜、仪器、图像处理、机器学习、聚合物材料、细胞生物学、分子生物学和基因组学。学员将能够利用高水平的技能、成就和通过研究项目开发的工程敏锐度，在工业界和学术界从事出色的职业。

The analysis of cells, or cytometry, is fundamentally important to many aspects of biological research. Flow cytometry has been the dominant cytometry technology since the 1960s because it is simple, high-throughput, and can be coupled with fluorescence activated cell sorting to separate specific cell populations. However, flow cytometry also has a number of limitations including (1) shear stress on the cell sample; (2) significant cell loss during processing; (3) difficulty in analyzing non-spherical cells, delicate cells, and cell clusters; (4) inability to assess sub-cellular structure and morphology. Many of these limitations could be addressed using fluorescence microscopy, but microscopy has the critical limitation of not being able to separate target cells, which in turn excludes many subsequent analyses, such as propagation to form clonal populations and single cell sequencing. Current technologies for isolating cells identified by microscopy involve physically transferring single cells using a micromanipulator, but this approach is difficult, low-throughput, and prone to contamination. The long-term vision of this research program is to develop microscopy-based technologies for cell separation, selective propagation, and selective single cell sequencing. Leveraging our recent work in this area, as well as advances in wide-field microscopy and image-processing, we believe our work will ultimately enable microscopy to compete with flow cytometry as a standard tool for cytometry, cell separation, and single cell analysis. This discovery grant will train HQPs at graduate, undergraduate, and post-doctoral levels. Trainees involved in the proposed research program will receive world-class training in an equitable, diverse, and inclusive environment that is dynamic and multi-disciplinary. Specific areas of research and training include optics, microscopy, instrumentation, image-processing, machine learning, polymer materials, cell biology, molecular biology, and genomics. Trainees will be able to leverage the high-level skills, accomplishments, and engineering acumen developed through their research projects into fantastic careers in industry and academia.