UC Irvine

Chapter 1. This chapter introduces fluorescence microscopy methods towards investigations in synthetic chemistry, with particular attention to polymer synthesis and polymer–catalyst dynamics, the topic of this dissertation. A powerful feature of fluorescence microscopy is its ability to obtain subensemble information about synthetic reactions that would otherwise be obscured by ensemble-averaging effects. A highlight of influential studies is provided herein, outlining how fluorescence microscopy was used to answer mechanistic questions in chemistry and inform on chemical dynamics.Chapter 2. Synthetic polymers, derived from transition metal-catalyzed polymerization, are some of the most ubiquitous materials used in contemporary society. Rates of these transition-metal catalyzed reactions are important to understand, given that there is direct relation between polymer structures and reaction rates; however, obtaining kinetic information at the single-catalyst and -polymer particle level is a challenge due to ensemble averaging present in traditional measurements. Kinetic information at the single-catalyst and -polymer particle level is vital for understanding the dynamic microenvironments of catalysts and developing a physical model for polymer particle growth. To address these gaps in knowledge, polymer structure modification techniques, crosslinking and spin coating, in tandem with fluorescence microscopy, were employed to observe the effect of polymer composition and morphology on molecular catalyst activity with single-polymer-particle sensitivity. These experiments exemplify the capabilities of fluorescence microscopy at the single polymer-particle level to elucidate a plausible model for particle growth during ROMP polymerization. A relation between polymerization rate and linear growth duration was found and provided a basis for interpreting particle growth rate data. Adapted with permission from Easter, Q. T.; Garcia IV, A.; Blum, S. A. Single-Polymer–Particle Growth Kinetics with Molecular Catalyst Speciation and Single-Turnover Imaging ACS Catal. 2019, 9, 3375−3383. Copyright 2019 American Chemical Society.

Chapter 3. The chemoselectivity of molecular catalysts underpins much of modern synthetic organic chemistry. However, little is known about the selectivity of individual catalysts because this single-catalyst-level behavior is hidden by the bulk catalytic behavior. Here, for the first time, the selectivity of individual molecular catalysts for two different reactions is imaged in real time at the single-catalyst level. This imaging is achieved through fluorescence microscopy paired with spectral probes that produce a snapshot of the instantaneous chemoselectivity of a single catalyst for either a single-chain-elongation or a single-chain-termination event during ruthenium-catalyzed polymerization. Superresolution imaging of multiple selectivity events, each at a different single-molecular ruthenium catalyst, indicates that catalyst selectivity may be unexpectedly spatially and time-variable. Copyright 2021 Wiley. Used with permission from Garcia IV, A.; Saluga, S. J.; Dibble, D. J.; López, P.; Saito, N.; Blum, S. A. Does Molecular Catalyst Selectivity Change with Time? Polymerization Imaged by Single-Molecule Spectroscopy. Angew. Chem. Int. Ed. 2021, 60, 1550−1555.

Chapter 4. The ability to directly observe chemical reactions at the single-molecule and single-particle level has enabled the discovery of behaviors otherwise obscured by ensemble averaging in bulk measurements. However powerful, a common restriction of these studies to date has been the absolute requirement to surface tether or otherwise immobilize the chemical reagent/reaction of interest. This constraint arose from a fundamental limitation of conventional microscopy techniques, which could not track molecules or particles rapidly diffusing in three dimensions, as occurs in solution. However, many chemical processes occur entirely in the solution phase, leaving single-particle/-molecule analysis of this critical area of science beyond the scope of available technology. Here, we report the first kinetics studies of freely diffusing and actively growing single polymer−particles at the single particle level freely diffusing in solution. Active-feedback single-particle tracking was used to capture three-dimensional (3D) trajectories and real-time volumetric images of freely diffusing polymer particles (D ≈ 10−12 m2 /s) and extract the growth rates of individual particles in the solution phase. The observed growth rates show that the average growth rate is a poor representation of the true underlying variability in polymer−particle growth behavior. These data revealed statistically significant populations of faster and slower-growing particles at different depths in the sample, showing emergent heterogeneity while particles are still freely diffusing in solution. These results go against the prevailing premise that chemical processes in freely diffusing solution will exhibit uniform kinetics. We anticipate that these studies will launch new directions of solution-phase, nonensemble-averaged measurements of chemical processes. Collaboration between the Blum Laboratory and the Welsher Laboratory at Duke University. Adapted with permission from Yu, D.; Garcia IV, A.; Blum, S. A.; Welsher, K. D. Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking J. Am. Chem. Soc. 2022, 9, 14698−14705. Copyright 2022 American Chemical Society.

Chapter 5. Control of polymer molecular weight is critical for tailoring structure−function properties; however, traditional molecular weight characterization techniques have limited ability to determine the molecular weight of polymers in real time without sample removal from the reaction mixture, with spatial resolution, and of insoluble polymers. In this work, a fluorescence lifetime imaging microscopy (FLIM) method was developed that overcomes these limitations. The method is demonstrated with polynorbornene and polydicyclopentadiene, polymers derived from ruthenium-catalyzed ring-opening metathesis polymerization (ROMP). The polymer Mw, ranging from 35 to 570 kg/mol as determined by gel permeation chromatography, was quantitatively correlated with the fluorescence lifetime. The revealed correlation then enabled time-resolved measurement of Mw during an ongoing ROMP reaction, requiring only 1 s per measurement (of a 45 μm × 45 μm polymer sample area), and provided spatial resolution, resulting in simultaneous characterization of polymer morphology. To provide the fluorescence signal, the initial reaction solutions contained a very low doping of a reactive norbornene monomer labeled with fluorescent boron dipyrromethene (BODIPY), such that 1 in every 107 monomers contained a fluorophore. The resulting FLIM visualization method enables the rapid determination of the molecular weights of growing polymers without removal from the reaction mixture and regardless of polymer solubility. Garcia IV, A.; Blum, S. A. Polymer Molecular Weight Determination via Fluorescence Lifetime. J. Am. Chem. Soc. 2022, 144, 22416−22420. Copyright 2022 American Chemical Society.

Chapter 6. This chapter summarizes unpublished projects including three research aims: 1) Investigating a universal approach to polymer molecular weight determination via fluorescence lifetime with non-covalently bound fluorescence dyes; 2) Developing FLIM based molecular weight determination through natively autofluorescent polymers and/or reaction systems; 3) Development of single-molecule FLIM methods to probe spatiotemporal heterogeneity in polymer molecular weight in real-time. These projects are inspired by the investigations from Chapter 5, where the overarching research goal is to address the current limitations to FLIM based molecular weight determination as well as provide information on molecular weight dispersity (PDI) in real-time.