High-resolution imaging methods are available for visualizing the spatial distribution of proteins and receptors on cell membranes, but these techniques do not provide functional information with regard to the activities of these proteins. Techniques capable of probing the functions of membrane proteins with high spatial and temporal resolution are limited, and much effort has been spent to develop such capabilities. The most notable and promising of these methods is the two-photon photolysis of photoactivable or “caged” molecules. Taking advantage of the nonlinear optical property of two-photon excitation, this technique can create a spatially focused un-caging volume that measures only a few hundred nanometers in width and height, as dictated by the far-field diffraction limit and the quadratic dependence of excitation on laser power. The availability of such a spatially and temporally selective method for probing cellular function and signaling dynamics is critical to understanding complex cellular and neuronal systems.
Despite the versatility offered by caged molecules, this approach is highly dependent on the availability of suitable photoactivable materials. The use of conventional chemically caged compounds suffers from a number of chemical drawbacks: (1) the design and synthesis of a suitable caged molecule is complex and time consuming, (2) the caging of large bioactive molecules such as peptides and proteins (e.g., cytokines) is difficult if at all possible, and (3) the un-caging of multiple stimuli simultaneously (e.g. for studying interactions among multiple signaling pathways) is often cumbersome. Because of these constraints, there are only a small number of caged molecules that have been developed over the past decades and an alternative technique for spatiotemporally delivering a stimulus to a biological system is needed.
USPTO Backgrounds
USPTO Backgrounds