Chemical reactions are defined by the change in the relative positions and bonding of nuclei in molecules. I have used femtosecond stimulated Raman spectroscopy (FSRS) to probe these transformations with structural specificity and high time precision revealing the mechanisms of two important classes of reactions; isomerization about an N=N bond and interfacial/intermolecular electron transfer.

Isomerization about a double bond is one of the simplest, yet most important, photochemical reactions. In contrast to carbon double bonds, nitrogen double bonds can react via two possible mechanisms; rotation or inversion. To determine which pathway is predominant, I studied an azobenzene derivative using both FSRS and impulsive stimulated Raman spectroscopy (ISRS). The FSRS experiments demonstrated that the photochemical reaction occurs concomitantly with the 700 fs non-radiative decay of the excited state; because no major change in N=N stretching frequency was measured, I surmised that the reaction proceeds through an inversion pathway. My subsequent ISRS experiments confirmed this hypothesis; I observed a highly displaced, low frequency, inversion-like mode, indicating that initial movement out of the Franck-Condon region proceeds along an inversion coordinate.

To probe which nuclear motions facilitate electron transfer and charge recombination, I used FSRS and the newly developed 2D-FSRS techniques to study two model systems, triphenylamine dyes bound to TiO₂ nanoparticles and a molecular charge transfer (CT) dimer. In the dye-nanoparticle system I discovered that charge separation persists much longer (> 100 ps) than previously thought by using the juxtaposition of the FSRS and transient absorption data to separate the dynamics of the dye from that of the injected electron. Additionally, I discovered that dye constructs with an added vinyl group were susceptible to quenching via isomerization. The CT dimer offered an opportunity to study a system in which charge recombination occurred on a shorter timescale (~10 ps). Here I found that impulsively excited coherent nuclear motion in the excited state survived for more than 5 ps. These long lived coherences enabled the performance of 2D-FSRS to identify the coupling and tuning modes of the conical intersection that connects the charge transfer excited state with the neutral ground state and therefore the nuclear motions responsible for charge recombination. Furthermore, these data demonstrate that the photochemical reactivity is not mediated by phase-space randomization but rather by specific, coherent, nuclear trajectories.

In this thesis I have demonstrated the power and utility of FSRS in probing condensed phase chemical reaction dynamics and provided a glimpse of the capabilities of 2D-FSRS. In general, 2D-FSRS provides a direct measure of the anharmonic coupling that controls reactivity. Looking forward, higher time resolution (broader band) 2D-FSRS will open up a whole new approach for characterizing transition states and energy transfer processes.

Organelle size control is an interesting problem in Biology. Organelles are often difficult to label or shaped in a way that is difficult to quantify. Chlamydomonas reinhardtii is a single celled alga with two equal length cilia. These cilia represent an ideal case study for organelle size control. Their size is easily quantifiable, as they are essentially one dimensional, only changing in length. The cilia are shed under acid shock, and completely regenerate within two hours. Measurement of length as a function of time during regeneration provides a quantitative insight into the regeneration process, and represents a crucial measurement required to fit predictions of length control models. But obtaining such measurements has proven extremely difficult because Chlamydomonas cells swim rapidly through the media. As a result, regeneration timecourse data usually is obtained at a population level using fixed samples. To enable high-throughput measurements of Chlamydomonas cilia length in living cells, we built a microfluidic device capable of trapping Chlamydomonas and solution exchange. Using this device, we found that instrinsic and extrinsic noise are present in the length control system. We were able to characterize the noise and assess its impact on biological function.

Novel methodologies for the molecular detection of human cancer have been advanced using microfabricated capillary electrophoresis devices as analytical platforms. These techniques enable direct and quantitative characterization of the unique biomolecular signature inherent to individual cancers and, through reduction of sample usage and analysis time, further the goal of routine genetic screening in the clinical setting.

The majority of the work detailed here applies a comparative sequencing technique known as Polymorphism Ratio Sequencing (PRS) to cancer detection with a focus on mutations in the mitochondrial DNA (mtDNA). First, a rigorous optimization of sample processing protocols was undertaken to improve PRS separations on a 96-lane microfabricated sequencing device. A modification of electrokinetic injection conditions has increased capillary success rates to nearly 100%, while the introduction of dynamic coatings and automated data processing has decreased analysis time by 75%. These optimized conditions were validated through a complete mtDNA sequence comparison of two unrelated individuals, uncovering 44 confirmed germline variations, eight of which were undetected in early PRS experiments. Further analysis of paired tumor and blood mtDNA from six individuals with lung and bladder cancer revealed three heteroplasmic somatic variants while uncovering 18 erroneous mutations identified in previous microarray analysis.

To establish potential clinical relevance, PRS was independently applied to a mitochondrial D-loop analysis of fourteen bladder cancer patients. A total of 21 somatic variations were identified, with seven patients harboring at least one mutation. Fifteen of these mutations were heteroplasmic, often occurring at low levels or problematic base locations inaccessible to conventional technologies. Where available, matched urine mtDNA was found to contain abundant populations of the mutant genotype, establishing the potential use of bodily fluids for noninvasive screening.

Finally, an integrated microdevice capable of PRS extension followed by inline purification and electrophoretic separation is presented. This device makes use of dual on-chip thermal cyclers and orthogonal Sanger extension primers to generate a complete set of PRS fragments prior to oligonucleotide-based capture and injection. Further integration with upstream sample processing steps, including single-cell capture and PCR amplification, is proposed, providing the framework for real-time mutant quantitation in microbiopsies, ultimately enabling full clinical integration.