Single molecule tracking studies of lower critical solution temperature transition behavior in poly(N-isopropylacrylamide)

In this thesis project, single molecule tracking, SMT, experiments in poly(n-isopropylacrylamide, pNIPAAm, were carried out using probe fluorophores that had partitioned into the polymer. When the pNIPAAm switches from expanded to collapsed at elevated temperatures, the free volume accessible to the diffusing molecules decreases and trajectories become more confined. The work presented here can be best understood when organized into two categories- SMT trajectory analysis techniques and SMT results in pNIPAAm. In the first category, four techniques for analyzing SMT data ??? confinement level analysis, time series analysis and statistical analysis of lateral diffusion, multistate kinetics, and a newly developed, radius of gyration evolution analysis ??? were compared using a set of sample fluorophore trajectories obtained from diffuse probe in surface-tethered pNIPAAm. The five SMT trajectories, ranging in length from 41 to 273 steps, were all successfully analyzed by all four techniques, provided two important criteria were met: enough steps to define the motion were acquired in the trajectory, generally on the order of 50 steps, and the fast and slow diffusion coefficients differed by at least a factor of 5. Beyond that, the four trajectory analysis methods studied provide partially confirmatory and partially complementary information. SMT data resulting from more complex physical behavior may well benefit from using these techniques in succession to identify and sort populations. In the second category of research, spatial and temporal heterogeneities in expanded and collapsed surface bound pNIPAAm films were studied by SMT experiments. Tracking data were analyzed using two methods involving radius of gyration (Rg) evolution and confinement level calculations. These two analysis techniques were used to elucidate the range of behaviors displayed by hundreds of single molecules, which exhibited complex diffusion. The main conclusions that were drawn from this work were: 1) small molecule probe behavior in pNIPAAm is dictated by the free volume within surface tethered chains, and the distribution of probe behavior can be used to gain nanometer-scale information about the state of the polymer brush at high and low temperatures, 2) confinement level calculations and radius of gyration evolution results show a larger degree of confinement at higher temperatures, measureable in higher percentage of confined steps in a trajectory, longer periods of confined events, and smaller area of confined zones