Laser-assisted substrate and cell membrane functionalization

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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.