Laser-assisted substrate and cell membrane functionalization

光学方法通过开启一系列探测和操纵分子事件的能力，彻底改变了现代细胞生物学。然而，仍然缺乏一种通用的，有效的和非侵入性的方法来标记观察到的单个细胞。目前的方法依赖于基因工程和光活化蛋白质，其产生复杂并且需要转化天然细胞和分子环境。* 我们最近开发了一种方法，通过光漂白细胞标记（CLaP），以非侵入性方式实现对单个细胞的即时，特异性标记。CLaP的简单性和多功能性是与广泛应用兼容的关键。CLaP利用激光照射在精确定义的细胞外区域产生高反应性分子，这些分子在活细胞的质膜上形成交联。使用CLaP，可以根据实验者在观察时选择的一系列标准，用激光标记单个细胞。标签不限于荧光模式，标记的细胞可以跟踪几天，分离和单独研究。本计划探索了CLaP成为以前技术上不可行的实验选项的具体领域，将该方法从我实验室的早期开发阶段应用于细胞生物学的工程新实现。我的短期计划的具体应用包括三个具体目标：*1）开发离体和体内双光子吸收CLaP。除了基因工程，电穿孔是用于标记体内单个细胞的唯一选择。我们希望证明CLaP是一种高精度，非侵入性的替代方法，不需要膜透化。2)创建基于CLaP的功能标签和分子递送。拴系功能性生物素化分子将使得能够进行仅操纵所选细胞的独特实验，例如，选择性转染或仅处理突触后细胞、快细胞、具有复杂乔木的细胞或接触的细胞。* 3)利用基于CLaP的瞬时细胞粘附。在异质群体中选择迁移细胞并操纵它们的运动代表了理解几个发育过程和许多疾病发病机制的基本机制所需的技术突破。基于图像的细胞选择标准的多功能性使得能够进行新的广泛的实验来解释细胞的背景或行为。CLaP技术允许分析基于其微环境和时空动力学选择的细胞，允许仅标记快速、大、圆、颗粒、孤立或遥远的细胞。这在细胞异质性发挥重要作用的领域尤其重要，包括发育、干细胞研究和神经生物学。

Optical approaches have revolutionized modern cell biology by opening a broad array of capabilities to probe and manipulate molecular events. Yet, a versatile, efficient and non-invasive approach to tag individual cells upon observations is still lacking. Current approaches rely on genetic engineering and photo-activatable proteins, which are complex to generate and require transformation of native cells and molecular environment. ***We have recently developed a method, Cell Labeling via Photobleaching (CLaP), that achieves instant, specific tagging of individual cells in a non-invasive manner. The simplicity and versatility of CLaP is key for compatibility with a broad range of applications. CLaP takes advantage of laser irradiation for generating highly reactive molecules in precisely defined extracellular regions that form crosslinks onto the plasma membrane of living cells. Using CLaP it is possible to tag individual cells with a laser, based on a wide array of criteria chosen by the experimenter at the time of observation. Tags are not restricted to fluorescent modalities, and labeled cells can be tracked for several days, isolated and individually studied.***The present program explores concrete areas where CLaP becomes an option to experiments that were not technically feasible before, taking the method from an early developmental stage in my lab to engineering new implementations for cellular biology. The particular applications of my short-term program comprise three specific aims:***1) Developing ex vivo and in vivo two-photon absorption CLaP. Beyond genetic engineering, electroporation is the only option used to label individual cells in vivo. We expect to demonstrate that CLaP is a high-precision, non-invasive alternative not requiring membrane permeabilization.***2) Creating CLaP-based functional tags and molecular delivery. Tethering functional biotinylated molecules will enable unique experiments manipulating only cells of choice, e.g., selectively transfecting or treating only post-synaptic cells, fast cells, cells with complex arbors or cells in contact.***3) Exploiting CLaP-based transient cell adhesion. Selecting migratory cells within a heterogeneous population and manipulating their movement represent a technological breakthrough needed for understanding several developmental processes and the fundamental mechanisms underlying the pathogenesis numerous diseases.******The versatility of image-based criteria for cell-selection enables a new wide range of experiments accounting for cellular context or behaviour. CLaP technology allows analyzing cells chosen based on their microenvironment and spatiotemporal dynamics, permitting to tag only fast, large, round, granular, isolated, or distant cells. This is particularly relevant in fields where cellular heterogeneity plays a major role, spanning development, stem cells research and neurobiology.