Micropatterning of Extracellular Matrix Proteins Using Microphoto-ablation Of Poly vinyl Alcohol (PVA) Monolayers

Available for licensure and commercial development is a micro-photoablation (µPA) method used as a micro-patterning technique to attach ECM proteins or other biological molecules to specified locations. Advantages of this photolytic technique are that it: (a) is stampless, (b) allows for flexible pattern generation to the submicron level, (c) allows for live cell fluorescence imaging, retains cell viability, and (d) allows the use of multiple proteins. The technique has demonstrated experimentally that micropatterning with live cell fluorescence imaging can be used to precisely visualize studying distinct cell-ECM interactions.

Applications of microlithography techniques into the study of cell biology aid in resolving cellular function as regulated by the interaction of cells with the extracellular matrix. Currently many techniques have used micro-contact patterning (µCP) to apply ECM proteins in distinct localized patterns. These techniques require the fabrication of silicone-based stamps to either "ink" proteins directly or indirectly onto a gold coated surface, limiting the user to a specified stamp shape and size. To bypass the necessity of a physical stamp the current technique provides submicron sized spots using a tunable multiphoton laser coupled to a confocal microscope to photo ablate hydrophilic poly vinyl alcohol (PVA) macro-molecular thin films. Through controlled photo ablation, PVA layers are locally removed allowing deposition of ECM proteins into distinct patterns. The use of ROI's produces a "virtual mask" that can be created in any shape or pattern and are easily modified. Unlike µCP techniques, micro-photoablation (µPA) allows live cell imaging of multiple fluorophores and is possible even with total internal reflection fluorescence (TIRF) microscopy. Therefore, micro-photo ablation (µPA) allows kinetic quantification of ECM-cell interactions. This technique that uses a macro-molecular thin film together with localized photo ablation allows the versatility to create protein spots of any size or shape easily on the same cover slip. Furthermore, this process can be repeated multiple times to directly conjugate different proteins to the same local region allowing the investigation of how single cells probe their surroundings to discern different ECM proteins.