Acquisition and analysis of steady-state and time-resolved fluorescence data for applications in materials science, bioanalytical chemistry, and super-resolution microscopy

Steady-state and time-resolved fluorescence techniques enjoy widespread applicability in domains ranging from biology to materials science owing to their extraordinary sensitivity and dynamic range. Among the most useful of these techniques is time-correlated, single-photon counting, which forms the basis of another: fluorescence lifetime imaging using stimulated emission depletion microscopy (FLIM-STED), which is used to obtain structural information on a subdiffraction-limited level (i.e., 40 nm or less). The high spatial resolution afforded by this technique is, however, accompanied by a reduction in the number of photons collected. Thus, its utility can only be exploited when meaningful information can be retrieved from sparse data sets. This retrieval requires the use of proper modeling and efficient analysis techniques. In this dissertation, several such techniques and their significance in super-resolution imaging are discussed in the context of extracting excited state fluorescence lifetime of one or more fluorophores. Probability-based, maximum-likelihood (ML) methods are compared with residual minimization (RM) methods in order to determine the limiting number of photons that are required to provide a meaningful analysis of the data. The ML methods are more robust and show considerable improvement over RM methods. The ML methods are further improved by implementing a Bayesian framework, where a nonuniform prior distribution of the parameters is included in the form of a Gaussian, an exponential, or a Dirichlet distribution. Two examples of the applications of the steady-state and time-resolved techniques are provided: the characterization of the properties of magnetic ionic liquids (MILs) and those of poly (3-hexylthiophene) (P3HT). MILs facilitate the solvent extraction of bioanalytes, e.g. DNA extraction from an aqueous solvent, with the help of an external magnetic field. The presence of paramagnetic ions, however, introduces several mechanisms of nonradiative quenching for the fluorescence of the label. Several MILs are screened to find a suitable candidate for DNA extraction using fluorescence spectroscopy. P3HT is used as the active donor layer of organic photovoltaics owing to their high photon-conversion efficiency. The structural details of the polymer aggregates of a thin film of P3HT exposed to electric filed are studied using steady-state and time-resolved anisotropy. Preferential orientations of the polymer backbone are observed if the thin film is exposed to an electric field during preparation