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Time Dependent Density Functional Theory offers a good accuracy/computational cost ratio among different methods used to predict the electronic structure for molecules of practical interest. The Coupled Electronic Oscillator (CEO) formalism was recently shown to accurately predict Nonlinear Optical (NLO) properties of organic chromophores when combined with Time Dependent Density Functional Theory. Unfortunately, CEO does not lend itself easily to interpretation of the structure activity relationships of chromophores. On the other hand, the Sum Over States formalism in combination with semiempirical wavefunction methods has been used in the past for the design of simplified essential states models. These models can be applied to optimization of NLO properties of interest for applications. Unfortunately, TD-DFT can not be combined directly with SOS because state-to-state transition dipoles are not defined in the linear response TD approach. In this work, a second order CEO approach to TD-DFT is simplified so that properties of double excited states and state-to-state transition dipoles may be expressed through the combination of linear response properties. This approach is termed the a posteriori Tamm-Dancoff approximation (ATDA), and validated against high-level wavefunction theory methods. Sum over States (SOS) and related Two-Photon Transition Matrix formalism are then used to predict Two-Photon Absorption (2PA) profiles and anisotropy, as well as Second Harmonic Generation (SHG) properties. Numerical results for several conjugated molecules are in excellent agreement with CEO and finite field calculations, and reproduce experimental measurements well.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

<p>The thesis consists of two main parts of nonlinear optical instrumentation development. </p> <p>Fluorescence-detected mid-infrared photothermal (F-PTIR) microscopy is demonstrated for sub-diffraction limited mid-infrared microspectroscopy of model systems and applied to probe phase transformations in amorphous solid dispersions. To overcome the diffraction limit in infrared imaging, a highly localized temperature-dependent photothermal effect is an attractive alternative indicator to infrared absorption. Photothermal atomic force microscopy infrared spectroscopy (AFM-IR) achieves nanometer resolution by monitoring heat caused expansion but only restricted on the surface. For 3D imaging, optically detected photothermal infrared (O-PTIR) combines an infrared laser with a visible probe source with to transduce photothermal refractive index changes (e.g., from changes in beam divergence or scattering). The sensitivity of O-PTIR is ultimately limited by the relatively weak dependence of refractive index with temperature, exhibiting changes of ~0.01% per oC. Fluorescence-detected photothermal mid-infrared (F-PTIR) spectroscopy (Fig. 1) is demonstrated herein to support 3D imaging with improved photothermal sensitivity. In F-FTIR, the sensitivity of fluorescence quantum efficiency to temperature change (~1-2% per oC) is used to transduce transient heat flux from localized IR absorption. The infrared spatial resolution of F-FTIR is defined by fluorescence microscopy and the thermal diffusivity of the sample instead of infrared wavelength. Initial F-PTIR proof of concept studies are described for microparticle assemblies of silica gel and polyethylene glycol, followed by applications of F-PTIR for analysis of localized composition within phase-separated domains induced by water vapor exposure of an amorphous solid dispersion (ritonavir in copovidone).</p> <p>Fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid diffusion mapping over the microsecond to millisecond time scale. Diffusion measurements are critical for molecular mobility assessment in cell biology, materials science and pharmacology. Fluorescence recovery after photobleaching (FRAP) is a well-known noninvasive optical microscopy method for measuring diffusion coefficients of macromolecules, such as proteins in cells and viscous solutions. However, conventional point-bleach FRAP is challenging to implement with multi-photon excitation and typically only supports diffusion analysis over millisecond time scales due to camera frame rate limitations. FRWP with patterned illumination addresses these limitations of FRAP by probing the fluorescence intensity changes while bleaching a comb pattern within a field of view (FoV). Fast-scanning of an ultrafast excitation beam distributes heat rapidly over multiple adjacent pixels, minimizing local heating effects that could complicate analogous diffusion measurements by point-bleach FRAP with multiphoton excitation. In FRWP, time-scales of the probed diffusion events are defined by a single line-pass time of a resonant scanning-mirror with a period of 125  s. In FRWP, the bleach pattern spans locations across the whole FoV, enabling diffusion mapping through image segmentation. More than a hundred bleaching and recovery events can be recorded during a single 10s measurement. Normal and anomalous diffusion of rhodamine-labeled bovine serum albumin (BSA) molecules was studied as a model system, with applications targeting rapid assessment of therapeutic macromolecule mobility within heterogeneous biological environments.</p>

<p>Space-based global navigation and precision timing systems are critical for modern infrastructure. Atomic clock technology has increased the precision of these systems so that they are viable for military operations, navigation, telecommunications, and finance. Advances in optical atomic clocks, based on optical frequencies, provide an opportunity for even more precise timing. Therefore, developments in chip-scale optical atomic clock technologies could lead to increased and more wide-spread application of this precision timing. One component of the optical atomic clock is the optical frequency comb which serves as an interface between optical and microwave frequencies. This thesis will cover experiments related to these optical frequency combs. A 2$\mu$m fiber laser was developed in order to test second harmonic devices required to stabilize an optical frequency comb. The laser was then employed to measure the operating wavelengths and efficiencies of non-linear devices. In addition, an analysis of the radio frequency instruments used to evaluate microwave outputs was conducted to determine whether a digital signal analyzer (oscilloscope) or an analog electronic spectrum analyzer provides more accurate results for optical frequency comb based experiments.</p>

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<p>The overall time and cost for a drug to go from the drug discovery to the consumer market is  significant,  showing  a  need  for  improved  drug  testing  and  discovery  methods.    Work  on nonlinear  optical  methods  for  both  small  active  pharmaceutical  ingredient  drug  formulation analysis and large biological therapeutic stability testing has been shown to improve testing times for formulation, stability and dissolution testing.  Herein, we review the existing and conventional approaches to address stability testing that the pharmaceutical industry uses, and how leveraging nonlinear optical (NLO) methods can improve the current challenges.  The specificity, sensitivity and low limit of detection of second harmonic generation is discussed in application to crystal formation in small-molecule active pharmaceutical ingredients.  The nonlinear optical methods second harmonic generation and two-photon excited ultraviolet fluorescence are directly compared to  ‘gold  standard’  powder  X-ray  diffraction,  which  is  commonly  used  for  measuring  crystal formation and growth of active pharmaceutical ingredients in amorphous solid dispersions.  In addition, the existing FRAP method (with multiple limitations) is improved upon with the ability to  perform  recovered  diffusion  coefficient  data  analysis  in  the  spatial  Fourier  domain.    The collective results discussed in this thesis are just a small subset of the total breadth of investigations marrying the new challenges in the pharmaceutical industry with the new NLO tools tailored to meet them</p>

Ionic self-assembled multilayers (ISAMs) can be formed by alternately dipping a substrate in anionic and cationic polyelectrolytes. Each immersion deposits a monolayer via electrostatic attraction, allowing for nanometer-scale control over film thickness. Additionally, ISAM films can be applied to arbitrary substrate geometries and can easily incorporate a variety of polymers and nanoscale organic or inorganic inclusions. The ISAM technique was used to tune and functionalize a rapid, sensitive fiber optic biosensor for textit{Brucella}, a family of bacteria that are detrimental to livestock and can also infect humans. The sensor was based on a turn-around point long period fiber grating (TAP-LPG). Unlike conventional LPGs, in which the attenuation peaks shift wavelength in response to environmental changes, TAP-LPGs have a highly sensitive single wavelength peak with variable attenuation. ISAMs were applied to a TAP-LPG to tune it to maximum sensitivity and to facilitate cross-linking of receptor molecules. Biotin and streptavidin were used to attach biotinylated hybridization probes specific to distinct species of textit{Brucella}. The sensor was then exposed to lysed cell cultures and tissue samples in order to evaluate its performance. The best results were obtained when using samples from textit{Brucella} infected mice, which produced a transmission change of 6.0 ± 1.4% for positive controls and 0.5 ± 2.0% for negative controls. While the sensor was able to distinguish between positive and negative samples, the relatively short dynamic range of the available fiber limited its performance. Attempts to fabricate new TAP-LPGs using a CO2 laser were unsuccessful due to poor laser stability. A second application of the ISAM technique was as a source of second-harmonic generation (SHG). SHG is a nonlinear optical process in which light is instantaneously converted to half its wavelength in the presence of intense electric fields. Localized surface plasmons (LSPs) in metal nanoparticles produce strong electric field enhancements, especially at sharp tips and edges, that can be used to increase SHG. Colloidally grown silver nanoprisms were deposited onto nonlinear ISAM films and conversion of 1064 nm Nd:YAG radiation to its 532 nm second-harmonic was observed. Little enhancement was observed when using nanoprisms with LSP resonance near 1064 nm due to their large size and low concentration. When using shorter wavelength nanoprisms, enhancements of up to 35 times were observed when they were applied by immersion, and up to 1380 times when concentrated nanoprisms were applied via dropcasting at high enough densities to broaden their extinction peak towards the excitation wavelength. A maximum enhancement of 2368 times was obtained when concentrated silver nanoprisms with LSP resonance around 900 nm were spincast with an additional layer of PCBS. / Doctor of Philosophy / Polyelectrolytes are long molecules composed of chains of charged monomers. When a substrate with a net surface charge is dipped into an oppositely charged polyelectrolyte solution, a single layer of molecules will be electrostatically deposited onto the substrate. Because the surface charge now appears to match the charge of the solution, no further deposition occurs. However, the process can be repeated by rinsing the substrate and immersing in a solution with the opposite charge. This technique forms ionic self-assembled multilayers (ISAMs), which can be assembled with nanometer-level control over thickness. The flexibility of polymer chemistry allows ISAMs to be formed from polyelectrolytes with a wide variety of properties. Additionally, the technique can easily incorporate other nanoscale materials, such as nanoparticles, clay platelets, and biological molecules, and has been investigated for applications ranging from dye-sensitized organic solar cells to drug delivery and medical implant coatings. This dissertation presents two applications of ISAM films. In one, ISAM films were used to tune and functionalize an optical biosensor for Brucella. Brucellosis primarily infects livestock, in which it causes significant reproductive problems leading to economic losses, but can also cause flu-like symptoms and more serious complications in humans. A rapid, sensitive test for Brucella is required to monitor herds and adjacent wild carriers, such as elk and bison. Optical biosensors, which operate by detecting changes due to the interaction between light and the stimulus, could satisfy this need. Long period fiber gratings (LPGs) are periodic modulations induced in the core of an optical fiber that cause transmitted light to be scattered at a resonant wavelength, resulting in attenuation. Conventional LPGs respond to changes in strain, temperature, or external refractive index by shifting their resonant wavelength. When special conditions are met, an LPG may exhibit a turn-around point (TAP), where dual peaks coalesce into a single peak with a constant wavelength but variable attenuation depth. TAP-LPGs are more sensitive than ordinary LPGs, and could be developed into inexpensive sensors with single-wavelength light sources and detectors. In this work, ISAMs were deposited onto an LPG to tune it near its TAP. Segments of single-stranded DNA, called hybridization probes, that were specific to individual species of Brucella were attached to the ISAM film before the sensor was exposed to lysed bacterial cultures. It was found that the sensor could distinguish between Brucella and other types of bacteria, but was less successful at distinguishing between Brucella species. The project was limited by the available TAP-LPGs, which had less dynamic range than those used in prior work by this group. Attempts were made to establish a new supply of TAP-LPGs by fabrication with a CO2 laser, but these efforts were unsuccessful due to poor laser stability. The second project discussed in this dissertation investigated ISAM films as a source of second-harmonic generation (SHG), a nonlinear optical process in which light is converted to half its fundamental wavelength in the presence of intense electric fields. Nonlinear ISAMs were constructed by choosing a polyelectrolyte with a hyperpolarizable side group in which SHG can occur. The SHG efficiency was increased by factors of several hundred to several thousand by the addition of silver nanoprisms. Metal nanoparticles can produce strong electric field enhancements, especially at their tips and edges, when incident light causes resonant collective oscillations in their electrons called localized surface plasmons (LSPs). It was found that while silver nanoprisms whose LSP resonant wavelength matched the fundamental wavelength were too dilute to produce noticeable enhancement, better results could be obtained by depositing shorter wavelength nanoprisms at sufficient density to broaden their extinction peak via interparticle interactions. The best enhancement observed was for a sample where concentrated silver nanoprisms with LSP resonance around 900 nm were dropcast onto an ISAM film and coated with an additional polymer layer, resulting in 2368 times more SHG than the plain ISAM film.

Terahertz (THz) spectroscopy is a powerful tool that uses ultrashort pulses of light to study the properties of materials on picosecond time scales. THz light can be generated through a variety of methods. In our lab, we generate THz through the process of optical rectification in nonlinear optical (NLO) organic crystals. THz light can be used to study several phenomena in materials, such as spin precession, electron acceleration, vibrational and rotational motion. The work presented in this dissertation is divided into two parts: (1) the generation of THz light and (2) applications of THz light. The first portion of this work shows how THz light is generated, with an emphasis on the generation through optical rectification. We also show how to improve the generation of THz light by creating heterogenous multi-layer structures with yellow organic THz generation crystals. Additionally, we show that crystals used for THz generation can also be used to generate second-harmonic light. In the second half of this work, we show that THz light can be used to study the vibrational motion of molecular systems. We model how resonant vibrational modes in a fluorobenzene molecule can be excited with a multi-THz pump to transfer energy anharmonically to non-resonant modes. We also show that we can use two-dimensional (2D) THz spectroscopy to excite infrared-active vibrational modes and probe Raman-active modes in a CdWO4 crystal to obtain a nonlinear response. We show that the nonlinear response is due to anharmonic coupling between vibrational modes and we can quantify the relative strengths of these anharmonic couplings, which previously was only accessible through first-principles calculations.