Measurement of normal and anomalous diffusion of dyes within protein structures fabricated via multiphoton excited cross-linking

We demonstrate microscale spatial and chemical control of diffusion within protein matrixes created through the use of nonlinear multiphoton excited photochemistry. The mobility of fluorescent dyes of different mass and composition within controlled cross-linked environments has been measured using two-photon excited fluorescence recovery after photobleaching (FRAP). The diffusion times for several rhodamine and sulforhodamine dyes within these fabricated structures were found to be approximately 3-4 orders of magnitude slower than in free solution. The precise diffusion times can be tuned by varying the laser exposure during the fabrication of the matrix, and the diffusion can be correlated with the mesh size determined by TEM and Flory-Rehner analysis. We find that the hydrophobic Texas Red dyes (sulforhodamines) exhibit diffusion that is highly anomalous, indicative of a strong interaction with the hydrophobic cross-linked protein matrix. These results suggests the use of these cross-linked protein matrixes as ideal model systems in which to systematically study anomalous diffusion. Finally, the diffusion can be tuned within a multilayered protein matrix, and this in conjunction with slow diffusion also suggests the use of these structures in controlled release applications.