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，使现代细胞生物学发生了革命性的变化。然而，一种通用的、高效的、非侵入性的方法来根据观察标记单个细胞仍然缺乏。目前的方法依赖于基因工程和可光激活的蛋白质，这些蛋白质的产生很复杂，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(CLAP)进行细胞标记，以非侵入性的方式实现对单个细胞的即时、特定标记。CLAP的简单性和多功能性是与广泛应用程序兼容的关键。CLAP利用激光照射在精确定义的胞外区域产生高活性分子，这些分子在活细胞的质膜上形成交联键。通过使用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。标签并不局限于荧光模式，标记的细胞可以被跟踪几天，分离和单独研究。*本项目探索了CLAP成为实验选项的具体区域，这些实验以前在技术上是不可行的，将该方法从我实验室的早期开发阶段带入设计细胞生物学的新实现。我的短期计划的具体应用包括三个具体目标：*1)开发体外和体内双光子吸收CLAP。除了基因工程，电穿孔是用来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、非侵入性的替代方案，不需要膜透过性。*2)创建基于CLAP的功能标签和分子递送。系留功能性生物素化分子将使仅操纵所选细胞的独特实验成为可能，例如，选择性地仅转染或治疗突触后细胞、快速细胞、具有复杂支架的细胞或接触的细胞。*3)利用基于CLAP的瞬时细胞黏附。选择异质群体中的迁移细胞并操纵它们的运动，是理解多种疾病的几个发育过程和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考虑细胞背景或行为的新的实验范围得以实现。CLAP技术允许分析根据微环境和时空动态选择的细胞，只允许标记快速、大型、圆形、颗粒状、孤立或远处的细胞。这在细胞异质性发挥主要作用的领域尤其相关，这些领域横跨发育、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.