The leading cause of death in patients who survive subarachnoid hemorrhage (SAH) is stroke as a result of cerebral arterial vasospasm1. Such vasospasms involve a vasoactive response, but they remain enigmatic and no clinical treatment has proven effective in prevention or reduction2. Arteries remodel in response to diverse mechanical loads and chemical factors. Following SAH, the surrounding vasculature is exposed to a radically altered chemo-mechanical environment. It is our hypothesis that chemical stimuli associated with the formation of an extravascular blood clot dominates the maladaptive growth and remodeling response early on, thus leading to important structural changes. However, it is not clear which of the many chemical factors are key players in the production of vasospasm. Before an accurate picture of the etiology of vasospasm can be produced, it is imperative to gain a better understanding of the non-pathogenic cerebral vasculature. In particular, the rabbit basilar artery is a well established model for vasospasm. However, surprisingly little is known about the mechanical properties of the rabbit basilar artery. Using an in vitro custom organ culture and mechanical testing device, acute and cultured basilar arteries from male White New Zealand specific pathogen free rabbits underwent cyclic pressurization tests at in vivo conditions and controlled levels of myogenic tone. Sections of basilar arteries were imaged for collagen fiber orientation at 0, 40 and 80 mmHg at in vivo stretch conditions using nonlinear optical microscopy. The nonlinear stress-strain curves provide baseline characteristics for acute and short-term culture basilar arteries. The active and passive testing creates a framework for interpreting the basal tone of arteries in our culture system. Nonlinear optical microscopy second harmonic generation provides unique microstructural information and allows imaging of live, intact arteries while maintaining in vivo geometries and conditions. Collagen fibers were found to be widely distributed about the axial direction in the adventitial layer and narrowly distributed about the circumferential direction in the adventitial layer. The quantified collagen fiber angles within the artery wall further support the development of accurate mathematical models.

The leading cause of death in patients who survive subarachnoid hemorrhage (SAH) is stroke as a result of cerebral arterial vasospasm1. Such vasospasms involve a vasoactive response, but they remain enigmatic and no clinical treatment has proven effective in prevention or reduction2. Arteries remodel in response to diverse mechanical loads and chemical factors. Following SAH, the surrounding vasculature is exposed to a radically altered chemo-mechanical environment. It is our hypothesis that chemical stimuli associated with the formation of an extravascular blood clot dominates the maladaptive growth and remodeling response early on, thus leading to important structural changes. However, it is not clear which of the many chemical factors are key players in the production of vasospasm. Before an accurate picture of the etiology of vasospasm can be produced, it is imperative to gain a better understanding of the non-pathogenic cerebral vasculature. In particular, the rabbit basilar artery is a well established model for vasospasm. However, surprisingly little is known about the mechanical properties of the rabbit basilar artery. Using an in vitro custom organ culture and mechanical testing device, acute and cultured basilar arteries from male White New Zealand specific pathogen free rabbits underwent cyclic pressurization tests at in vivo conditions and controlled levels of myogenic tone. Sections of basilar arteries were imaged for collagen fiber orientation at 0, 40 and 80 mmHg at in vivo stretch conditions using nonlinear optical microscopy. The nonlinear stress-strain curves provide baseline characteristics for acute and short-term culture basilar arteries. The active and passive testing creates a framework for interpreting the basal tone of arteries in our culture system. Nonlinear optical microscopy second harmonic generation provides unique microstructural information and allows imaging of live, intact arteries while maintaining in vivo geometries and conditions. Collagen fibers were found to be widely distributed about the axial direction in the adventitial layer and narrowly distributed about the circumferential direction in the adventitial layer. The quantified collagen fiber angles within the artery wall further support the development of accurate mathematical models.

A detailed understanding of the corneal biomechanical response is an important prerequisite to understanding corneal diseases such as keratoconus and for placing the empirical equations used in refractive surgery on a physical basis. We have assembled a combined nonlinear optical microscopy (NLOM) and optical coherence microscopy (OCM) imaging system to simultaneously capture coregistered volumetric images of corneal morphology and biochemistry. Fudicial markers visible in the OCM volume enabled the calculation of strains for multiple depth layers in rabbit cornea. The results revealed a depth dependent strain distribution, with smaller strains in the anterior stroma and larger strains in the posterior stroma. The stress-strain curves can be grouped readily by depth into three groups: anterior (~20%), transitional mid (~40%), and posterior (~40%). Cross-sectional images of collagen lamellae, visible in NLOM, showed inhomogeneous collagen structure and its response to intraocular pressure along the anterior-posterior direction. The inhomogeneities correlate well with the noted heterogeneous corneal mechanical properties. The combined NLOM-OCM system can measure corneal microstructure and mechanical response uniquely, thus providing a microstructural understanding of corneal response to changes of collagen structure.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Stimulated Raman spectroscopy (SRS) is a nondestructive tool for biochemical characterization of tissues. The aims were: 1) To evaluate the ability of SRS and Spontaneous Raman spectroscopy (SpRS) to differentiate among sound, demineralized and remineralized bovine enamel by phosphate and carbonate ratio (P/C-Ratio); and 2) To determine the correlation between the outcomes of transverse microradiography (TMR: Integrated mineral loss (ΔZ) and lesion depth) and P/C-Ratio. Material and Methods: Thirty, 5×5×2-mm ground and polished bovine enamel blocks were prepared. The surface was divided into 3 equal areas. Each area was chemically demineralized (demin) by Carbopol demineralized solution for 0 (Sound-Demin), 24 (24h-Demin), and 48h (48h-Demin), respectively. Then, specimens were sectioned for TMR analysis, and the remaining one part of each specimen was remineralized (remin) for 15days using a pH-cycling model (Sound-Remin, 24hD-Remin=24h-Demin and remineralization, 48hD-Remin = 48h-Demin and remineralization). Demin and remin groups were scanned to obtain P/C-Ratio by SpRS and SRS. SRS was further scanned from 0 (surface) up to 100 µm into the dentine at 10-µm intervals. Remineralized specimens were sectioned for TMR analysis. Wilcoxon signed-rank tests were used to compare between TMR and SpRS/SRS. Spearman correlation coefficients were used to correlate among TMR, SpRS, and SRS. A 5-percent significance level was used for each test. Results: As demin time increased, both ΔZ and lesion depth were increased. After remineralization, both values were decreased. There were significant differences between demine and remin groups and between demin times. For SpRS, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin (p ≤ 0.001). The 24h-Demin had significantly larger values than 48h-Demin (p = 0.048). Sound-Remin had larger P/C-Ratio than 24hD-Remin (p = 0.316) and 48hD-Remin (p = 0.015). 24hD-Remin was larger than 48hD-Remin (p = 0.269). 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin (p ≤ 0.001). 48hD-Remin had significantly larger P/C-Ratio than 48h-Demin (p ≤ 0.001). For SRS, at surface (0 µm), for demin group, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin (p = 0.020) and 48h-Demin (p = 0.032). 24h-Demin had larger value than 48h-Demin; but no significant difference (p = 0.117). Among remin groups, Sound-Remin was not statistical significance different for 24hD-Remin (p = 0.172) and 48hD-Remin (p = 0.134). However, 24hD-Remin was smaller; but not statistical significance different from 48hD-Remin (p = 0.688). At deeper levels (10 µm to 100µm), it was found that 1) After demineralization, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin at 0 µm to 20 µm, and 80 µm to 100µm; Sound-Demin had significantly larger P/C-Ratio than 48h-Demin; and no statistical significance differences were found among Sound-Demin and 24h-Demin, 24h-Demin and 48h-Demin. 2) After remineralization, no statistical significance differences were found among Sound-Remin, 24hD-Remin, and 48hD-Remin. 3) Sound-Demin had significantly larger P/C-Ratio than Sound-Remin at 0 µm ,10 µm, 20 µm; and no statistical significance differences were found at levels deeper than 30 µm. 4) No statistical significance differences were found between 24h-Demin and 24hD-Remin from 0 µm to 70µm; and 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin from 80 µm to 100 µm. 5) No statistical significance differences were found between 48h-Demin and 48hD-Remin. For correlation, moderate correlation was found between SpRS demineralized/remineralized groups and ΔZ, and between SpRS demineralized groups and lesion depth. Conclusion: SpRS and SRS have the potential to quantify demineralization through calculation of the phosphate and carbonate ratio. In addition, SpRS can detect the change of remineralization. A nondestructive caries detection approach using SpRS and SRS would be beneficial in clinical practice.

Histology has long recognized the intimate link between structure and function. Over centuries histologists have utilized an assortment of optical microscopy techniques to elucidate functional attributes of tissues through investigating tissue architecture. This thesis includes developments in the field of nonlinear optical microscopy for use in histology and pathology. The main contributions focused on the study of fibrillar collagen in the extracellular matrix (ECM) with polarization-dependent second harmonic generation (P-SHG) microscopy and the study of harmonophore-stained cellular nuclei with third harmonic generation (THG) microscopy. The P-SHG microscopy technique, “polarization-in, polarization-out” (PIPO), was developed to accurately determine the second-order polarization properties of thin tissue sections. The polarization instrumentation was implemented into a nonlinear optical microscope and a custom fitting algorithm extracted ratios of the second-order nonlinear susceptibility elements at every pixel of an obtained image. Hierarchical organization, at every level of structure, can contribute significantly to the macroscopic second-order polarization properties of fibrillar collagen in the ECM and quantifiable differences between the various tissue architectures were observed. A framework was developed, based on the collagen hierarchical organization, to interpret the submicron polarization properties of various tissues. Complimentary to the P-SHG study of connective tissue, the structure of hematoxylin and eosin (H&E) stained nuclei was revealed by THG microscopy. Imaging the 3D organization of nuclei was possible using the inherent optical sectioning provided by nonlinear microscopy. The origin of THG was investigated with spectrally- and temporally-resolved measurements, as well as the THG ratio method. A rather complex situation involving multiple dye complexes was revealed. The structure of dye aggregates was investigated with THG PIPO microscopy. The techniques of PIPO and harmonophore-stained harmonic generation microscopy show great potential for ultimately furthering understanding of tissue structure and function. H&E stained tissue investigations with THG microscopy has applications as a tool for cancer diagnostics. PIPO can elucidate the symmetry and organization of materials beyond tissues, including starch, nanowires, and protein crystals. In pathology, the developed collagen framework has strong implications, as collagen is recognized as playing a more active role in a number of diseases including idiopathic pulmonary fibrosis, wound repair, and tumour development and progression.

Histology has long recognized the intimate link between structure and function. Over centuries histologists have utilized an assortment of optical microscopy techniques to elucidate functional attributes of tissues through investigating tissue architecture. This thesis includes developments in the field of nonlinear optical microscopy for use in histology and pathology. The main contributions focused on the study of fibrillar collagen in the extracellular matrix (ECM) with polarization-dependent second harmonic generation (P-SHG) microscopy and the study of harmonophore-stained cellular nuclei with third harmonic generation (THG) microscopy. The P-SHG microscopy technique, “polarization-in, polarization-out” (PIPO), was developed to accurately determine the second-order polarization properties of thin tissue sections. The polarization instrumentation was implemented into a nonlinear optical microscope and a custom fitting algorithm extracted ratios of the second-order nonlinear susceptibility elements at every pixel of an obtained image. Hierarchical organization, at every level of structure, can contribute significantly to the macroscopic second-order polarization properties of fibrillar collagen in the ECM and quantifiable differences between the various tissue architectures were observed. A framework was developed, based on the collagen hierarchical organization, to interpret the submicron polarization properties of various tissues. Complimentary to the P-SHG study of connective tissue, the structure of hematoxylin and eosin (H&E) stained nuclei was revealed by THG microscopy. Imaging the 3D organization of nuclei was possible using the inherent optical sectioning provided by nonlinear microscopy. The origin of THG was investigated with spectrally- and temporally-resolved measurements, as well as the THG ratio method. A rather complex situation involving multiple dye complexes was revealed. The structure of dye aggregates was investigated with THG PIPO microscopy. The techniques of PIPO and harmonophore-stained harmonic generation microscopy show great potential for ultimately furthering understanding of tissue structure and function. H&E stained tissue investigations with THG microscopy has applications as a tool for cancer diagnostics. PIPO can elucidate the symmetry and organization of materials beyond tissues, including starch, nanowires, and protein crystals. In pathology, the developed collagen framework has strong implications, as collagen is recognized as playing a more active role in a number of diseases including idiopathic pulmonary fibrosis, wound repair, and tumour development and progression.

A thorough understanding of pharmaceutical and therapeutic products and materials is important for an improved quality of life. By probing the complex behaviors and properties of these systems, new insights can allow for a better understanding of current treatments, improved design and synthesis of new drug products, and the development of new treatments for various health conditions. Often, the impact of these new insights are limited by current technology and instrumentation and by the methods in which existing data is processed. Additionally, current standards for characterization of pharmaceuticals and therapeutics are time-consuming and can delay the timeline in which these products become available to the consumer. By addressing the limitations in current instrumentation and data science methods, faster and improved characterization is possible.<div><br></div><div>Development and improvement in optical instrumentation provides potential solutions to the current limitations of characterization methods by conventional instrumentation. Limitations in speed can be addressed through the use of nonlinear optical (NLO) methods, such as second harmonic generation (SHG) and two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy, or by linear methods such as fluorescence recovery after photobleaching (FRAP). For these methods, a high signal-to-noise ratio (SNR) and a nondestructive nature decrease the overall sample size requirements and collections times of these methods. Furthermore, by combination of these optical techniques with other techniques, such as thermal analysis (e.g. differential scanning calorimetry (DSC)), polarization modulation, or patterned illumination, the collection of more complex and higher quality data is possible while retaining the improved speed of these methods. Thus, this modified instrumentation can allow for improved characterization of properties such as stability, structure, and mobility of pharmaceutical and therapeutic products.<br></div><div><br></div><div>With an increase in data quantity and complexity, improvements to existing methods of analysis, as well as development of new data science methods, is essential. Machine learning (ML) architectures and empirically validated models for the analysis of existing data can provide improved quantification. Using the aforementioned optical instrumentation, auto-calibration of data acquired by SHG microscopy is one such method in which quantification of sample crystallinity is enabled by these ML and empirical models. Additionally, ML approaches utilizing generative adversarial networks (GANs) are able to improve on identification of data tampering in order to retain data security. By use of GANs to tamper with experimentally collected and/or simulated data used in existing spectral classifiers, knowledge of adversarial methods and weakness in spectral classification can be ascertained. Likewise, perturbations in physical illumination can be used to ascertain information on classification of real objects by use of GANs. Use of this knowledge can then be used to prevent further data tampering or by improving identification of data tampering.<br></div>

Dans cette thèse, nous proposons l'utilisation d'un nouveau type de fibre à cristal photonique, la fibre Kagomé à coeur creux, pour la livraison d'impulsions ultra-courtes en endoscopie non linéaire. Ces fibres permettent la livraison d'impulsions sans distorsion sur une large bande spectrale, avec un faible bruit de fond, grâce à la propagation dans le cœur creux. Nous avons résolu le problème de la résolution spatiale, à l'aide d'une microbille en silice, insérée dans le cœur de la fibre Kagomé. Nous avons développé un système d'imagerie compacte, qui utilise un tube piézo-électrique pour le balayage du faisceau, un système achromatiques de microlentilles et une fibre Kagomé double gaine, spécialement conçue pour l'endoscopie. Avec ce système, nous avons réussi à imager des tissus biologiques, à l'extrémité distale de la fibre (endoscopie), en utilisant des différentes techniques tels que TPEF, SHG et CARS, un résultat qui ne trouve pas d'égal dans la littérature actuelle. L'intégration dans une sonde portable (4,2 mm de diamètre) montre le potentiel de ce système pour de futures applications en endoscopie multimodale in-vivo. / In this thesis, we propose the use of a novel type of photonic crystal fiber, the Kagomé lattice hollow core fiber, for the delivery of ultra-short pulses in nonlinear endoscopy. These fibers allow undistorted pulse delivery, over a broad transmission window, with minimum background signal generated in the fiber, thanks to the propagation in a hollow-core. We solved the problem of spatial resolution, by means of a silica micro-bead inserted in the Kagomé fiber large core. We have developed a miniature imaging system, based on a piezo-electric tube scanner, an achromatic micro-lenses assembly and a specifically designed Kagomé double-clad fiber. With this system we were able to image biological tissues, in endoscope modality, activating different contrasts such as TPEF, SHG and CARS, at the distal end of the fiber, a result which finds no equal in current literature. The integration in a portable probe (4.2 mm in diameter) shows the potential of this system for future in-vivo multimodal endoscopy.