The current work investigated the stimulation induced spatial response of the cerebral microvascular network by reconstruction of the 3D microvascular morphology from in vivo two photon fluorescence microscopy (2PFM) volumes using an automated, model based tracking algorithm. In vivo 2PFM imaging of the vasculature in the forelimb representation of the primary somatosensory cortex of alpha-chloralose anesthetized rats was achieved via implantation of a closed cranial window, and intravascular injection of fluorescent dextran. The dilatory and constrictory responses of the cerebral microvascular network to functional stimulation were heterogeneous and depended on resting vascular radius and response latency. Capillaries experienced large relative dilations and constrictions, but the larger vessel absolute volume changes dominated the overall network cerebral blood volume change.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

The current work investigated the stimulation induced spatial response of the cerebral microvascular network by reconstruction of the 3D microvascular morphology from in vivo two photon fluorescence microscopy (2PFM) volumes using an automated, model based tracking algorithm. In vivo 2PFM imaging of the vasculature in the forelimb representation of the primary somatosensory cortex of alpha-chloralose anesthetized rats was achieved via implantation of a closed cranial window, and intravascular injection of fluorescent dextran. The dilatory and constrictory responses of the cerebral microvascular network to functional stimulation were heterogeneous and depended on resting vascular radius and response latency. Capillaries experienced large relative dilations and constrictions, but the larger vessel absolute volume changes dominated the overall network cerebral blood volume change.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

Nonlinear Optical Microscopy (NLOM) has been shown to be a valuable tool for noninvasive imaging of complex biological systems. An effective approach for multicolor molecular microscopy is simultaneous excitation of multiple fluorophores by broadband sub-10-fs pulses. This dissertation will discuss the development of two spectral imaging systems using the principles of nonlinear optical microscopy for pixel-by-pixel spectral segmentation of multiple fluorescent spectra. The first spectral system is reliant on a fiber-optic cable to transmit fluorescent signal to a spectrometer, while the second is based on a spectrometer with an aberration-corrected concave grating that is directly coupled to the microscope. A photon-counting, 16-channel multianode photomultiplier tube (PMT) is used for both systems. Custom software developed in LabVIEW controls multiple counter cards as well as a field-programmable gate array (FPGA) for 1 Hz acquisition of 256x256x16 spectral images. Biological specimen consisting of multicolor endothelial cells and zebrafish will be used for experimental verification. Results indicate successful spectral segmentation of multiple fluorophores with a decrease in signal-to-noise ratio in the FPGA-based imaging system.

This thesis develops a novel photopatterning strategy for biomimetic scaffolds that enables spatial and biochemical control of engineered cellular architectures, such as the microvasculature. Intricate tools that allow for the three dimensional (3D) manipulation of biomaterial microenvironments will be critical for organizing cellular behavior, directing tissue formation, and ultimately, developing functional therapeutics to treat patients with critical organ failure. Poly(ethylene glycol) (PEG) based hydrogels, which without modification naturally resist protein adsorption and cellular adhesion, were utilized in combination with a two-photon laser patterning approach to covalently immobilize specific biomolecules in custom-designed, three-dimensional (3D) micropatterns. This technique, known as two-photon laser scanning lithography (TP-LSL), was shown in this thesis to possess the capability to micropattern multiple different biomolecules at modular concentrations into a single hydrogel microenvironment over a broad range of size scales with high 3D resolution. 3D cellular adhesion and migration were then explored in detail using time-lapse confocal microscopy to follow cells as they migrated along micropatterned tracks of various 3D size and composition. Further, in a valuable modification of TP-LSL, images from the endogenous microenvironment were converted into instructions to precisely direct the laser patterning of biomolecules within PEG-based hydrogels. 3D images of endogenous microvasculature from various tissues were directly converted into 3D biomolecule patterns within the hydrogel scaffold with precise pattern fidelity. While tissue engineers have previously demonstrated the formation of vessels through the encapsulation of endothelial cells and pericyte precursor cells within PEG-based hydrogels, the vessel structure had been random, uncoordinated, and therefore, ultimately non-functional. This thesis has utilized image guided TP-LSL to pattern biomolecules into a 3D structure that directs the organization of vessels to mimic that of the endogenous tissue vasculature. TP-LSL now stands as a valuable tool to control the microstructure of engineered cellular architectures, thereby providing a critical step in the development of cellularized scaffolds into functional tissues. Ultimately, this thesis develops new technologies that advance the field of regenerative medicine towards the goal of engineering viable organs to therapeutically treat the 18 patients who die every day waiting on the organ transplant list.

Esta tesis describe el diseño, la fabricación y la caracterización óptica de sistemas plasmónicos resonantes capaces de confinar y aumentar campos de luz en la escala manométrica. En primer lugar, se utilizaron modelos numéricos 3D para diseñar diferentes geometras de nanoestructuras plasmónicas acopladas, a través del cálculo de la respuesta óptica de su campo lejano y cercano. Sobre la base de estas simulaciones se fabricaron las nanoestructuras por litografía de haz electrónico. Se puso especial énfasis en el aumento de la resolución y la optimización de la reproducibilidad de parámetros críticos como la forma de las partículas y el gap entre ellas. Por último, se empleó espectroscopía de campo lejano combinada con espectroscopía de luminiscencia inducida por dos fotones (TPL) para sondar la respuesta óptica local de las geometrías optimizadas. Hemos centrado nuestra atención en diferentes tipos de estructuras metálicas: dímeros, antenas con gap, conjuntos finitos de partículas en cadenas y en forma de estrella. Los dímeros tienen una fuerte amplificación del campo en su gap nanométrico por el acoplamiento en campo cercano de sus resonancias plasmonicas dipolares. Análogamente, antenas con gap, formadas por dos barras de oro adyacentes que soportan resonancias multipolares, pueden acoplar de manera eficiente la luz y concentrarla en volúmenes pequeños. Se ha demostrado que cadenas finitas de partículas son buenos candidatos para guiar la luz a través de secciones transversales por debajo de la longitud de onda y aquí demostramos que también se pueden utilizar como nanolentes capaces de concentrar la luz en su extremo. La distribución del campo cercano en conjuntos de partículas de oro en forma de estrella presenta una fuerte dependencia con la polarización del campo incidente que puede ser explotada para dirigirse dinámicamente a nano-objetos. La espectroscopía de campo lejano de conjuntos de dímeros y de cadenas finitas de partculas se comparó con la espectroscopía de TPL. Nuestro principal resultado es mostrar que la TPL es preferentemente sensible a los campos locales, permitiendo evaluar características espectrosc ópicas que no podrían resolverse de otro modo. A fin de superar las limitaciones de las medidas de conjuntos, en una segunda etapa se dedicó un considerable esfuerzo a construir y optimizar un montaje óptico para medir la señal de TPL de estructuras únicas. El uso de la micro-espectroscopía de TPL permitió obtener mapas espectrales de los modos de antenas aisladas con resolución espacial. Como se predijo mediante cálculos, hemos sido capaces de visualizar directamente, en la resonancia, la señal de TPL amplificada dentro del gap. Nuestros resultados muestran cómo las medidas de TPL pueden compararse directamente con la distribución de la cuarta potencia del campo local calculado. Mediante el análisis de la evolución de la señal de TPL en función de la longitud de onda incidente en el gap y en las extremidades de la antena tenemos más conocimiento sobre el mecanismo físico detrás de la resonancia de la antena. Finalmente, la microscopía de TPL se utilizó para sondar el campo cercano para diferentes orientaciones de la polarización lineal incidente sobre los conjuntos de partículas en forma de estrella. Se demuestra que, a diferencia del espectro de dispersión, la distribución de TPL en la estructura depende drásticamente del estado de polarización incidente. Nuestro estudio aporta una contribución significativa al campo de la óptica de plasmones, proponiendo nuevas geometrías para confinar de manera eficiente los campos ópticos a la escala nanometrica, aportando un profundo conocimiento sobre el uso de micro-espectroscopa de TPL como sonda óptica local. Nuestros resultados tendrán importancia en aplicaciones tales como espectroscopía mejorada, biosensores y la interacción luz-materia, donde se necesita evaluar el campo experimentado por una pequeña cantidad de materia cercana a la nanoestructura. / This thesis describes the design, fabrication and the optical characterization of plasmon-resonant systems able to confine and enhance light fields down to the sub-wavelength scale. Extensive 3D numerical modeling was first used to design different geometries of coupled plasmonic nanostructures through the calculation of their far-field and near-field optical response. On the basis of simulations, the nanostructures were fabricated by e-beam lithography and thin film deposition. Special efforts were devoted to increasing the resolution and optimizing the reproducibility of critical parameters such as particle shape and interparticle gaps. Finally, far-field spectroscopy combined with two-photon induced luminescence (TPL) spectroscopy was used to probe the local optical response of the optimized architectures. We focused our attention on different families of structures: metal dimers, bar antennas, finite chains of nanoparticles and star-like particle arrangements. Particle dimers feature strong field enhancements in their sub-wavelength gap due to near-field coupling of their dipolar localized plasmon resonances. Based on the same physics, gap antennas, formed by two adjacent gold bars supporting multipolar resonances can efficiently couple to propagating light and concentrate it into tiny volumes. While finite particle chains were previously shown by other authors to be good candidates to guide light through subwavelength cross-sections, we show here that they can also be used as efficient nanolenses able to concentrate light at their extremity. Finally, the near-field distribution in star-like arrangements of gold nanoparticles exhibits a strong dependence with the incident field polarization which can be exploited for dynamical optical addressing of nano-objects. We have compared the far field spectroscopy of large ensembles of dimers and finite chains to TPL spectroscopy. Our main result is to show that TPL is preferentially sensitive to local fields and that it enables the assessment of spectroscopic features which cannot be resolved otherwise. In order to overcome the limitations of measurements on large ensembles a considerable effort was dedicated to mounting and optimizing an optical set-up enabling TPL measurement of single structures. Using the developed TPL micro-spectroscopy, spatially resolved spectral mode mapping on single resonant gap-antennas was achieved. As predicted by calculations, we were able to directly visualize at resonance the strongly enhanced TPL signal within the gap. Our results show how TPL scans can be directly compared with the convoluted distribution of the fourth power of the calculated local mode field. By monitoring the evolution with the incident wavelength of the TPL signal within the gap and at the antenna extremities we got further insight in the physical mechanism behind the buildup of the antenna’s resonance. Finally, TPL microscopy was used to probe the local fields under different orientations of the incident linear polarization near star-like arrangement of gold disks. It is shown that, unlike the scattering spectrum, the TPL distribution over the structure is found to depend drastically on the incident polarization state. Our study brings a significant contribution to the field of Plasmon optics by proposing novel geometries able to efficiently confine optical fields down to the nanometric scale, but also by providing deep insight into the use of TPL microspectroscopy to probe their local optical response. Our findings are foreseen to be important in applications such as enhanced spectroscopy, bio-sensing and enhanced light-matter interaction, where one needs to assess the actual field experienced by small amounts of matter.

High quality YbxY1-xAG (0≤x≤1) single crystals were grown by using the Czochralski method. The structural properties of YbxY1-xAG (0≤x≤1) single crystals were also investigated using the EXAFS method. Additionally, for the first time, EXAFS results were compared with XRD results. Moreover, without essential lattice structure transformation, YbxY1-xAG crystals can be found due to the varying of doping concentration in XRD measurements. However, EXAFS measurements indicated that the local fine structural variation around the Yb3+ ion depends on the Yb3+ concentration. This work also studies the two-photon luminescence spectra of a Yb3+ doped YAG crystals. To study the spectral performance of Yb:YAG crystal, intense green light (centered at about 544nm) was generated by the crystal upon excitation using a 973nm InGaAs LD pump source. The luminescence spectra were obtained for various Yb concentrations and the emission intensity were plotted versus Yb3+ concentration. The green emission light attributed to Yb3+ two-photon transition.

In this study, the characteristic properties of the ultrafast laser exhibit sufficiently in the application of RFOBIC and two-photon UV fluorescence. This laser can be used to measure photonic components with fast responding speed due to the ultrashort pulse and broad bandwidth which is RF bandwidths of greater than 1.8THz. we have demonstrated the use of a frequency-doubled femtosecond optical parametric oscillator in generating two-photon excitation that is equivalent to ultraviolet(UV) light with wavelength less than 300 nm. This capability allows observation of some amino acids and enables excitation that is only possible with wavelength in UVB range(290 nm-320 nm)