Single Cell Optical Manipulations

生物样品的表征在很大程度上依赖于显微镜，其中响应于各种刺激，分子探针和令人印象深刻的对比试剂列表可用于识别和标记感兴趣的活细胞。这些方法，例如转染编码荧光蛋白的基因、使用膜可渗透染料或抗体标记，需要细胞标记物的先验知识或使用精心设计的报告构建体。然而，仅仅基于视觉检查或使用图像处理算法，就有可能从显微镜视野内的数千个对应物中区分出表现出不同生物学特性的细胞。事实上，大量的关键生物过程是由细胞特征决定的，这些特征只能在直接显微镜观察时进行评估。这种视觉表型通常具有重要的和充分理解的分子和生物学意义，但缺乏用于实验研究的实用生物化学标记。例如，细胞运动在正常胚胎发育、伤口愈合和免疫反应中至关重要。细胞形状与生长、分裂和死亡相关，因为细胞骨架特性决定细胞分化，这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和细胞与细胞的接触在化学运动、神经功能和免疫反应中至关重要。 我的NSERC发现计划的总体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识别，标记，操纵，捕获和扩增细胞。我提出的实现这一目标的工具是基于我实验室最近的技术发展，例如通过光漂白进行细胞标记（CLaP），它可以在观察时标记单个细胞。CLaP使用激光照射交联活细胞质膜上的生物素和荧光链霉亲和素缀合物，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，从技术的不同能力中获利，并扩展其应用。1)开发基于单细胞CLaP的基因递送。CLaP可以与基于病毒的技术相结合，用激光在显微镜视野中选择待转染的单个细胞。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序列结合到培养物中的单个细胞。使用激光照射将这些DNA标签结合到质膜上，并且仅结合到选定的单个细胞上。测序后将鉴定标记细胞。3)使用CLaP从视觉表型中获得新的细胞群。我将使用细胞运动性作为基础，创造新的细胞系，这将允许揭示通常隐藏在大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.