Single Cell Optical Manipulations

生物样品的表征在很大程度上依赖于显微镜，在显微镜下，分子探针和一系列令人印象深刻的造影剂可以用来识别和标记感兴趣的活细胞。这些方法，如编码荧光蛋白的基因的转染，使用膜渗透染料或抗体标记，需要事先了解细胞标记或使用复杂的报告结构。然而，仅基于肉眼检查或使用图像处理算法，就有可能从显微镜领域内的数千个对应细胞中区分出表现出不同生物学特性的细胞。事实上，大量关键的生物过程是由细胞特性决定的，这些特性只能通过直接显微镜观察来评估。这种视觉表型通常具有重要的和众所周知的分子和生物学意义，但缺乏用于实验研究的实用生化标记。例如，细胞运动在正常胚胎发育、伤口愈合和免疫反应中是必不可少的。细胞形状与生长、分裂和死亡有关，因为细胞骨架特性决定细胞分化，而细胞分化在组织形态发生和疾病中至关重要。此外，细胞与化学信号的距离以及细胞与细胞之间的接触对化学运动、神经功能和免疫反应都是至关重要的。我的NSERC发现计划的首要目标是专注于技术开发，允许基于只能通过显微镜成像获得的视觉信息来识别、标记、操作、捕获和扩展细胞。我提出的实现这一点的工具是基于我实验室的最新技术发展，例如光漂白细胞标记(CLAP)，它可以根据观察对单个细胞进行标记。CLAP使用激光照射将生物素交联到活细胞的质膜上，并用荧光链霉亲和素结合物以不同的方式与它们相互作用。在接下来的五年里，我提出了三个目标，包括开发CLAP，从该技术的不同能力中获利，并扩大其应用。1)发展基于单细胞CLAP的基因传递。CLAP可以与基于病毒的技术相结合，用激光选择显微镜领域中要转基因的单个细胞。2)生成并测试用于单细胞测序的CLAP寡核苷酸条形码。CLAP可用于将预定义的生物素化DNA序列与培养中的单个细胞结合。这些DNA标签使用激光照射结合到质膜上，并且只结合到选定的单个细胞上。标记的细胞将在测序后进行鉴定。3)使用CLAP从视觉表型中获得新的细胞群体。我将以细胞运动性为基础，创造新的细胞系，以揭示通常隐藏在大型细胞系中的分子机制。

Characterization of biological samples relies heavily on microscopy where, in response to various stimuli, molecular probes and an impressive list of contrast reagents can be used to identify and label live cells of interest. These methods, such as transfection of genes encoding fluorescent proteins, the use of membrane-permeable dyes or antibody labeling require prior knowledge of cellular markers or use of elaborate reporter constructs. However, based solely on visual inspection or using image processing algorithms, it is possible to distinguish cells which exhibit distinct biological properties from among thousands of counterparts within a microscopy field. Indeed, a large number of critical biological processes are determined by cellular characteristics that can only be assessed upon direct microscopy observation. Such visual phenotypes often have major and well-understood molecular and biological implications but lack practical biochemical markers for experimental research. For example, cell movement is essential in normal embryonic development, wound healing and immune responses. Cell shape is associated with growth, division and death, as cytoskeletal properties determine cell differentiation, essential in tissue morphogenesis and disease. Furthermore, the position of cells regarding distance to chemical cues and cell-to-cell contact are critical in chemokinesis, neural function and immune responses. The overarching goal of my NSERC Discovery program is focused on technology developments that allow identifying, tagging, manipulating, capturing and expanding cells based on visual information that can only be obtained by microscopy imaging. The tools I propose to achieve this are based on recent technological developments from my lab, such as Cell Labeling via Photobleaching (CLaP) which can tag individual cells upon observation. CLaP uses laser irradiation for crosslinking biotin on the plasma membrane of living cells and fluorescent streptavidin conjugates to interact with them in different ways. For the next five years I propose three objectives that comprise the exploitation of CLaP, profiting from different capacities of the technology and extending its applications. 1) To develop single cell CLaP-based gene delivery. CLaP can be combined with virus-based technology to select with a laser the individual cells in a microscopy field to be transfected. 2) To generate and test CLaP oligonucleotide barcodes for single cell sequencing. CLaP can be used to bind predefined biotinylated DNA sequences to individual cells in culture. These DNA tags are bound to the plasma membrane using laser illumination and only to selected individual cells. Labeled cells will be identified after sequencing. 3) To use CLaP to derive novel cell populations from visual phenotypes. I will use cell motility as the basis to create novel cell lines that will allow uncovering of molecular mechanism normally hidden in large cell ensembles.