Zum ersten Mal konnte die kohärente Erzeugung von ultrakurzen Pulsen mit Feld stärken im MV/cm Bereich mit einem Spektralbereich von 0.1-30 THz im organischen Kristall DSTMS. Kohärente mehrdimensionale Terahertzspektroskopie (CMTS) hat sich zu einer wichtigen Methode zur Untersuchung der niederenergetischen Anregungen von Halbleitern and deren kohärenter Dynamik entwickelt. Eine neuartige CMTS Methode mit drei phasenstarren, zueinander zeitverzögerten Terahertzpulsen wurde entwickelt. Sie beruht auf der kollinearen Wechselwirkung der Pulse mit der Probe, sodass verschiedene Ordnungen des nichtlinearen Signals in gleicher Richtung emittiert werden und deshalb gleichzeitig gemessen werden können. Amplitude und Phase des nichtlinearen Signals können durch elektro-optisches Abtasten vermessen werden, wodurch die zeitliche Entwicklung der kohärenten Wechselwirkungen in Echtzeit untersucht werden kann. CMTS erlaubt zusätzlich die eindeutige Zerlegung des nichtlinearen Signals in die verschiedenen nichtlinearen Ordnungen in der jeweiligen mehrdimensionalen Frequenzdomäne. Die nichtlineare, nicht-resonante Antwort zweier undotierter Halbleiter, des Ferroelektrikums Lithiumniobat (LiNbO3) und Indiumantimonids (InSb) kann mit dieser neuartigen Methode untersucht werden. In LiNbO3 wird das nichtlineare Signal durch einen Femtosekunden nichtlinearen Verschiebestrom (SC) hervorgerufen. SC wird durch die gebrochene Inversionssymmetrie des Kristalls in Verbindung mit einer ultraschnellen Dephasierung der feldinduzierten, kohärenten interband-Polarisation hervorgerufen. Die Dephasierung der interband-Polarisation erlaubt das Tunneln von Elektronen vom Valenzband in das Leitungsband. In InSb wird das kohärente Signal durch sowohl zwei-Phonen als auch zwei-Photonen interband-Anregungen erzeugt. Die impulsive Anregung einer kohärenten zwei-Phononen Polarisation wird durch das große Übergangsdipolmoment von InSb verstärkt, was zu deutlich größeren Amplituden der Polarisation als im linearem Regime führt. / For the first time, the coherent generation of ultrashort MV/cm field pulses with a spectrum covering the frequency range 0.1-30 THz is demonstrated in the organic crystal DSTMS. Coherent multidimensional terahertz spectroscopy (CMTS) has become a prominent technique for, e.g., driving low-energy excitations in semiconductors and monitoring their coherent dynamics. A novel CMTS technique using three phase-locked inter-delayed THz pulses is implemented. It relies on a collinear interaction of the pulses with a sample, so that different contributions to the nonlinear signal are emitted in the same direction, and thus can be measured all at once. Phase-resolved detection by electro-optic sampling allows for measuring amplitude and absolute phase of the nonlinear signal, thereby enabling to investigate the evolution of coherent interactions between quantum excitations in real time. In CMTS, the nonlinear signal is dissected into the distinct nonlinear contributions in the corresponding multidimensional frequency domain. This novel technique is applied to study the nonlinear off-resonant response of two undoped bulk semiconductors, the wide-bandgap ferroelectric lithium niobate (LiNbO3) and the narrow-bandgap indium antimonide (InSb). In LiNbO3, the nonlinear signal is generated by a femtosecond nonlinear shift current (SC), a distinctive characteristic of the bulk photovoltaic effect. The SC stems from the lack of inversion symmetry and the ultrafast dephasing of the field-induced interband coherent polarization due to a sufficiently high decoherence rate, which enables tunneling of electrons from the valence to the conduction band. In InSb, the nonlinear signal is caused by the coherent response on both the two-phonon and two-photon interband excitations. The impulsive generation of the two-phonon coherent polarization is enhanced by the large interband transition dipole of InSb, resulting in much larger polarization amplitudes than in the regime of linear response.

Im Rahmen dieser Arbeit wurden fluoreszierende Sphingomyelin-Analoga zu Studium der sauren Sphingomyelinase (ASM) synthetisiert. Ausgehend von L-Serin wurde ein Sphingosin-Derivat mit natürlicher Stereochemie dargestellt. Anschließend wurde mittels Phosphorodichloridat-Chemie eine Aminoethylphosphat-Gruppe installiert. Zweifache Fluoreszenzmarkierung ergab Sonden mit der Fähigkeit zu Förster-Resonanzenergietransfer (FRET). Diese wurden als Substrate der ASM akzeptiert und erlaubten die Verfolgung der Enzymaktivität in vitro. Durch die Analyse der photophysikalischen Eigenschaften der Fluorophore wurde das allgemeine Konzept der Phasentrennungs-gestützten Signalverstärkung (PS) abgeleitet. Dieses Konzept wurde erfolgreich bestätigt durch die Synthese einer 30-mal leistungsfähigeren zweiten Generation der FRET-Sonde. Ein homogener Assay wurde entwickelt, der die Quantifizierung der ASM-Aktivität erlaubte. Unter Verwendung von gereinigter rekombinanter humaner ASM, HeLa-Zelllysaten oder Lysaten von murinen embryonalen Fibroblasten (MEFs) als Enzymquelle wurde ausschließlich unter den von der ASM bevorzugten Bedingungen eine vollständige und spezifische Hydrolyse der Sonde beobachtet. Des Weiteren erlaubte die Sonde die Detektion relativer Unterschiede der Aktivität der ASM in kultivierten MEFs mittels Fluoreszenzmikroskopie mit Zweiphotonenanregung (2PE). / Fluorescent sphingomyelin analogues have been synthesized to probe the acid sphingomyelinase (ASM). Starting from L-serine, a sphingosine with natural stereochemistry was synthesized. Subsequently, phosphorodichloridate chemistry was used to install an aminoethyl phosphate moiety. Dual fluorescent labeling afforded probes capable of Förster resonance energy transfer (FRET). They were recognized as substrates of ASM and allowed for monitoring of the enzyme’s activity in vitro. Through analysis of the fluorophores’ photophysical properties, the general concept of partition aided amplification of a FRET probe’s signal (PS) was developed. This concept was successfully confirmed by the synthesis of a second-generation probe with 30-fold improved response. A homogenous assay was developed, which allowed for a quantitation of ASM activity. Using either purified recombinant human ASM, or lysates of HeLa cells or mouse embryonic fibroblasts (MEFs) as an enzyme source, complete and specific cleavage was observed exclusively under conditions preferred by ASM. Furthermore, the probe enabled the detection of relative levels of ASM activity in cultivated MEFs using fluorescence microscopy with two-photon excitation (2PE).

Optics are a powerful probe of chemical structure that can often be linked to theoretical predictions, providing robustness as a measurement tool. Not only do optical interactions like second harmonic generation (SHG), single and two-photon excited fluorescence (TPEF), and infrared absorption provide chemical specificity at the molecular and macromolecular scale, but the ability to image enables mapping heterogeneous behavior across complex systems such as biological tissue. This thesis will discuss nonlinear and linear optics, leveraging theoretical predictions to provide frameworks for interpreting analytical measurement. In turn, the causal mechanistic understanding provided by these frameworks will enable structurally specific quantitative tools with a special emphasis on application in biological imaging. The thesis will begin with an introduction to 2nd order nonlinear optics and the polarization analysis thereof, covering both the Jones framework for polarization analysis and the design of experiment. Novel experimental architectures aimed at reducing 1/f noise in polarization analysis will be discussed, leveraging both rapid modulation in time through electro-optic modulators (Chapter 2), as well as fixed-optic spatial modulation approaches (Chapter 3). In addition, challenges in polarization-dependent imaging within turbid systems will be addressed with the discussion of a theoretical framework to model SHG occurring from unpolarized light (Chapter 4). The application of this framework to thick tissue imaging for analysis of collagen local structure can provide a method for characterizing changes in tissue morphology associated with some common cancers (Chapter 5). In addition to discussion of nonlinear optical phenomena, a novel mechanism for electric dipole allowed fluorescence-detected circular dichroism will be introduced (Chapter 6). Tackling challenges associated with label-free chemically specific imaging, the construction of a novel infrared hyperspectral microscope for chemical classification in complex mixtures will be presented (Chapter 7). The thesis will conclude with a discussion of the inherent disadvantages in taking the traditional paradigm of modeling and measuring chemistry separately and provide the multi-agent consensus equilibrium (MACE) framework as an alternative to the classic meet-in-the-middle approach (Chapter 8). Spanning topics from pure theoretical descriptions of light-matter interaction to full experimental work, this thesis aims to unify these two fronts. <br>

Indiana University-Purdue University Indianapolis (IUPUI) / Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.