Use of steady-state and time-resolved fluorescence spectroscopy as a tool to investigate photophysics of biologically and environmentally relevant systems

Steady-state and time-resolved fluorescence spectroscopy are among the most widespread and powerful tools in the study of physical, chemical and biological systems. In this thesis, we discuss the use of these technologies to study range of important processes occurring on timescales from femtoseconds (10-15 s) to nanoseconds (10-9 s). In particular, we employ the techniques of time-correlated single photon counting and fluorescence upconversion, which are described in detail in subsequent chapter. The physical problems that we address with these technologies are: solvation dynamics in various systems, especially proteins; the use of ionic liquids for the hydrolysis of cellulose; and stereoselective photophysics in chiral ionic liquids

Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the Xand Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging

Analytical and data strategy for continuous downstream manufacturing

As advances emerge in developing continuous biomanufacturing processes, there is an increased need to deploy PAT tools to characterize, monitor, and control key quality attributes and a criticality to have a data infrastructure to support the immense amount of information being generated. While the desire for these tools exists in traditional batch processing, in a continuous operation, these become a requirement to ensure consistent product quality and enable proactive approaches in maintaining performance. The ultimate goal is to deploy PAT tools to reliably provide real-time information on product and process impurities throughout the entire operation. However, in its current state, there is a reliance on a mixture of inline, at-line, and offline technologies. By identifying the time criticality of CQAs, efforts can be focused on where to prioritize real-time measurements or instead, quicker or more automated testing for a subset of analytics. This work describes the application of this approach in the development of small-scale, compact in-line UV instruments to measure real-time protein concentration and in the integration of an automated sampling system with at-line and offline instrumentation for in-process impurity characterization. Introduction of these PAT tools add to the complexity of the data infrastructure as it introduces requirements for platforms capable of supporting spectral data, chemometric model deployment, spectral instrument management, and time-alignment of discrete data. With the vast amount of information produced in a continuous environment, interface and analysis tools need to be developed so that any end-user can digest data into a format that easily allows them to gain insight into an ongoing batch. This work will highlight the data architecture of the continuous platform, with a focus on software tools selected for aggregation and real-time data visualization. The capabilities of these software packages were demonstrated through a proof-of-concept study using single-pass tangential flow filtration (SPTFF) as a model unit operation, which allowed integration of continuous, spectral, and discrete data. These tools allowed scientists to go from viewing real-time data across multiple, equipment-specific software to one consolidated interface, which in turn reduced time spent in compiling data for analysis and reporting. In addition, advanced capabilities of deploying model predictive control in SPTFF were demonstrated to show the application of a closed loop process control in continuous manufacturing

Dynamic Solvation in Phosphonium Ionic Liquids: Comparison of Bulk and Micellar Systems and Considerations for the Construction of the Solvation Correlation Function, C(t)

Dynamic solvation of the dye coumarin 153 is studied in a phosphonium ionic liquid:  hexadecyltributylphosphonium bromide, [(C4)3C16P+][Br-]. It forms micelles in water, and the bulk also exists as a liquid under our experimental conditions. This system permits a comparison with an imidazolium ionic liquid studied earlier, which also formed micelles in water (J. Phys. Chem. A 2006, 110, 10725−10730). We conclude that our analysis of the comparable situation in a phosphonium liquid is not as definitive as we had proposed earlier, i.e., that the majority of the early-time solvation arises from the organic cation. Part of the difficulty in performing this analysis is most likely due to the amount of water that is associated with the micelle. In the course of this work, we have focused on the calculation of the solvation correlation function, C(t), and investigated how it depends upon the methods with which the “zero-time” spectrum is constructed