The development of super-resolution microscopy made it possible to surpass the diffraction limit of optical microscopy, enabling researchers to gain a nanometer scale understanding of cellular structures. While many applications have benefited from standard super-resolution microscopy, gaps remained making high-speed dynamic imaging in live cells impossible. To address this problem, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed. This methodology enables the nanometer imaging of dynamic cell processes within live cells, the evaluation of subcellular structural information, the capacity to derive three-dimensional information from two-dimensional images within rotationally symmetric structures, and the interrogation of novel questions regarding the transport dynamics of macromolecules in a variety of cellular structures. Here, I have described the theory and method behind the current iteration of SPEED microscopy that we have developed and validated via Monte Carlo simulation. Further, a detailed description of how we have further developed SPEED microscopy to derive structural information within the nuclear pore complex as well as how SPEED has been applied to evaluate the export kinetics of mRNA. / Biology

The mechanisms responsible for ice propagation on surfaces undergoing dropwise condensation have been determined and characterized. Based on experimental data acquired non-invasively with high speed quantitative microscopy, the freezing process was determined to occur by two distinct mechanisms: inter-droplet and intradroplet ice crystal growth. The inter-droplet crystal growth mechanism was responsible for the propagation of the ice phase between droplets while the intra-droplet crystal growth mechanism was responsible for the propagation of ice within individual droplets. The larger scale manifestation of these two mechanisms cooperating in tandem was designated as the aggregate freezing process. The dynamics of the aggregate freezing process were characterized in terms of the substrate thermal di usivity, the substrate temperature, the free stream air humidity ratio, and the interfacial substrate properties of roughness and contact angle, which were combined into a single surface energy parameter. Results showed that for a given thermal di usivity, the aggregate freezing velocity increased asymptotically towards a constant value with decreasing surface temperature, increasing humidity, and decreasing surface energy. The inter-droplet freezing velocity was found to be independent of substrate temperature and only slightly dependent on humidity and surface energy. The intra-droplet freezing velocity was determined to be a strong function of substrate temperature, a weaker function of surface energy, and independent of humidity. From the data, a set of correlational models were developed to predict the three freezing velocities in terms of the independent variables. These models predicted the majority of the measured aggregate, inter- and intra-droplet freezing velocities to within 15%, 10%, and 35%, respectively. Basic thermodynamic analyses of the inter- and intra-droplet freezing mechanisms showed that the dynamics of these processes were consistent with the kinetics of crystal growth from the vapor and supercooled liquid phases, respectively. The aggregate freezing process was also analyzed in terms of its constituent mechanisms; those results suggested that the distribution of liquid condensate on the surface has the largest impact on the aggregate freezing dynamics.

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