Generation of micropatterned substrates using micro photopatterning.

Micro photopatterning (microPP) has been developed to rapidly test and generate different patterns for extracellular matrix adsorption without being hindered by the process of making physical stamps through nanolithography techniques. It uses two-photon excitation guided through a point-scanning confocal microscope to locally photoablate poly(vinyl) alcohol (PVA) thin films in user-defined computer-controlled patterns. PVA thin films are ideal for surface blocking, being hydrophilic substrates that deter protein adsorption and cell attachment. Because gold substrates are not used during microPP, all live-cell fluorescent-imaging techniques including total internal reflection fluorescence microscopy of GFP-linked proteins can be performed with minimal loss of fluorescence signal. Furthermore, because microPP does not require physical stamps for pattern generation, multiple ECMs or other proteins can be localized within microns of each other. This unit details the setup of microPP. It also provides troubleshooting techniques.