Renewable forms of energy are becoming increasingly important as the world quickly depletes its current energy reserves, and rapidly increases the concentration of pollutants in our environment. Solar technology based on organic semiconductors provides a promising candidate to fulfill a portion of our future energy needs in an environmentally sustainable manner. Organic semiconductors are a collection of pi-conjugated small molecules and polymers which can be implemented in photovoltaic cells that are potentially quite low cost. Currently, however, their commercial applications are limited due to a relatively low efficiency in converting sunlight into usable power. The fundamental physics of such devices must be clarified if these materials are to compete with traditional inorganic solar cells. In this dissertation, two emerging experimental tools are implemented in investigations of the internal electric fields present within operating organic photovoltaic cells. The first set of investigations utilizes the vibrational Stark effect to quantify the electric fields which often form at the interfaces between two organic semiconducting materials. Such interfaces are at the heart of the photocurrent generation process in these devices and any electric fields formed crucially alter device performance. We quantitatively determine the interfacial field present in blends of poly(3-hexylthiophene) (P3HT) and phenyl-C61-butyric acid methyl ester (PCBM) and show that this field depends strongly on annealing conditions. Finally we discuss a correlation between this interfacial electric field, crystalinity and device performance. The second set of investigations take advantage of electric field induced second harmonic generation microscopy to examine the electric potential across active organic solar cells. We again investigate blends of PCBM and P3HT as well as poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6-diyl-alt-(2,1,3-benzothiadiazole)-4,7-diyl) (PSBTBT) and PCBM. In the former we find that the potential drop across the device shifts dramatically over time under illumination, while in the latter we find a nearly linear drop which remains constant through device operation. We then extend our examinations of PSBTBT:PCBM with EFISH by quantifying the extent of space charge accumulation throughout such devices. / text

Coherent Raman microscopy (CRM) is a powerful nonlinear optical imaging technique based on contrast via Raman active molecular vibrations. CRM has been used in domains ranging from biology to medicine to geology in order to provide quick, sensitive, chemical-specific, and label-free 3D sectioning of samples. The Raman contrast is usually obtained by combining two ultrashort pulse input beams, known as Pump and Stokes, whose frequency difference is adjusted to the Raman vibrational frequency of interest. CRM can be used in conjunction with other imaging modalities such as second harmonic generation, fluorescence, and third harmonic generation microscopy, resulting in a multimodal imaging technique that can capture a massive amount of data. Two fundamental elements are crucial in CRM. First, a laser source which is broadband, stable, rapidly tunable, and low in noise. Second, a strategy for image analysis that can handle denoising and material classification issues in the relatively large datasets obtained by CRM techniques. Stimulated Raman Scattering (SRS) microscopy is a subset of CRM techniques, and this thesis is devoted entirely to it. Although Raman imaging based on a single vibrational resonance can be useful, non-resonant background signals and overlapping bands in SRS can impair contrast and chemical specificity. Tuning over the Raman spectrum is therefore crucial for target identification, which necessitates the use of a broadband and easily tunable laser source. Although supercontinuum generation in a nonlinear fibre could provide extended tunability, it is typically not viable for some CRM techniques, specifically in SRS microscopy. Signal acquisition schemes in SRS microscopy are focused primarily on detecting a tiny modulation transfer between the Pump and Stokes input laser beams. As a result, very low noise source is required. The primary and most important component in hyperspectral SRS microscopy is a low-noise broadband laser source. The second problem in SRS microscopy is poor signal-to-noise (SNR) ratios in some situations, which can be caused by low target-molecule concentrations in the sample and/or scattering losses in deep-tissue imaging, as examples. Furthermore, in some SRS imaging applications (e.g., in vivo), fast imaging, low input laser power or short integration time is required to prevent sample photodamage, typically resulting in low contrast (low SNR) images. Low SNR images also typically suffer from poorly resolved spectral features. Various de-noising techniques have been used to date in image improvement. However, to enable averaging, these often require either previous knowledge of the noise source or numerous images of the same field of view (under better observing conditions), which may result in the image having lower spatial-spectral resolution. Sample segmentation or converting a 2D hyperspectral image to a chemical concentration map, is also a critical issue in SRS microscopy. Raman vibrational bands in heterogeneous samples are likely to overlap, necessitating the use of chemometrics to separate and segment them. We will address the aforementioned issues in SRS microscopy in this thesis. To begin, we demonstrate that a supercontinuum light source based on all normal dispersion (ANDi) fibres generates a stable broadband output with very low incremental source noise. The ANDi fibre output's noise power spectral density was evaluated, and its applicability in hyperspectral SRS microscopy applications was shown. This demonstrates the potential of ANDi fibre sources for broadband SRS imaging as well as their ease of implementation. Second, we demonstrate a deep learning neural net model and unsupervised machine-learning algorithm for rapid and automated de-noising and segmentation of SRS images based on a ten-layer convolutional autoencoder: UHRED (Unsupervised Hyperspectral Resolution Enhancement and De-noising). UHRED is trained in an unsupervised manner using only a single (“one-shot”) hyperspectral image, with no requirements for training on high quality (ground truth) labelled data sets or images.

No description available.

Microscopy techniques have been developed and refined over multiple decades, but innovation around single photon modalities has slowed. The advancement of the utility of information acquired, and minimum resolution available is seemingly reaching an asymptote. The fusion of light microscopy and well-studied nonlinear processes has broken through this barrier and enabled the collection of vast amounts of additional information beyond the topographical information relayed by traditional microscopes. Through nonlinear imaging modalities, chemical information can also be extracted from tissue. Nonlinear microscopy also can beat the resolution limit caused by diffraction, and offers up three-dimensional capabilities. The power of nonlinear imaging has been demonstrated by countless research groups, solidifying it as a major player in biomedical imaging. The value of a nonlinear imaging system could be enhanced if a reduction in size would permit the insertion into bodily cavities, as has been demonstrated by linear imaging endoscopes. The miniaturization of single photon imaging devices has led to significant advancements in diagnostics and treatment in the medical field. Much more information can be extracted from a patient if the tissue can be imaged in vivo, a capability that traditional, bulky, table top microscopes cannot offer. The development of new technologies in optics has enabled the miniaturization of many critical components of standard microscopes. It is possible to combine nonlinear techniques with these miniaturized elements into a portable, hand held microscope that can be applied to various facets of the biomedical field. The research demonstrated in this thesis is based on the selection, testing and assembly of several miniaturized optical components for use as a nonlinear imaging device. This thesis is the first demonstration of a fibre delivered, microelectromechanical systems mirror with miniaturized optics housed in a portable, hand held package. Specifically, it is designed for coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excitation fluorescence imaging. Depending on the modality being exploited, different chemical information can be extracted from the sample being imaged. This miniaturized microscope can be applied to diagnostics and treatments of spinal cord diseases and injuries, atherosclerosis research, cancer tumour identification and a plethora of other biomedical applications. The device that will be revealed in the upcoming text is validated by demonstrating all designed-for nonlinear modalities, and later will be used to perform serialized imaging of myelin of a single specimen over time.

La technique de spectroscopie basée sur la diffusion Raman Stokes spontanée est un procédé standard employé dans de nombreux domaines allant de la thermodynamique à la médecine, en passant par la science des matériaux. À la faveur d'un échange d'énergie inélastique, elle permet de déterminer les fréquences des vibrations moléculaires présentes dans un objet. On peut ainsi remonter à l'identification des molécules et ainsi caractériser l'objet d'étude sans utiliser de marqueur spécifique. Cette méthode est néanmoins affligée de défauts. Outre la présence d'un signal de fluorescence qui peut submerger la réponse Raman, le désavantage majeur est le long temps d'exposition que requière cette technique. Dans le cas d'étude d'échantillon biologique, cela proscris son usage pour des mesures de microspectroscopie : la cartographie spectrale d'objet microscopique. Afin de pallier ce problème, de nouvelles techniques ont été développées. C'est le cas de la spectroscopie employant la diffusion Raman anti-Stokes Cohérente (ou CARS pour Coherent Anti-Stokes Raman Scattering). Du fait de sa cohérence et de sa directivité le signal anti-Stokes affiche une intensité 10^5 to 10^6 fois plus importante que dans le cas de la diffusion Raman spontanée, ce qui permet alors d'abaisser le temps d'exposition à un niveau tolérable pour les objets biologiques lors d'une mesure de microspectroscopie. De plus, le caractère anti-Stokes du signal l'épargne de la contribution de la fluorescence. Pourtant, un défaut majeur limite encore l'utilisation de cette technique : le bruit de fond non résonant. Ce phénomène peut diminuer, voir noyer la contribution résonante qui porte l'information. Cette thèse a permis le développement de techniques CARS autorisant une réduction du bruit de fond non résonant. Pour ce faire un dispositif de spectroscopie CARS multiplex (M-CARS) en configuration copropagative a été construit. Ses capacités sont illustrées par des mesures spectrales d'échantillons minéral, végétal et biologique. À partir de ce système, il a été établi une méthode innovante permettant de discriminer le signal résonant du bruit non résonant en utilisant un champ électrique continu. Il est aussi démontré la mise en place d'un procédé qui a permis de mener la première mesure de microspectroscopie M-CARS en configuration contrapropagative sur un échantillon biologique. Cette configuration limite la collecte du signal à l'objet d'étude, empêchant ainsi l'acquisition du signal résonant et non résonant issu du solvant, principal responsable du bruit de fond non résonant lors d'une mesure CARS en configuration copropagative. / The spectroscopy technique based on spontanée Raman Stokes scattering is a standard process used in many fields spanning from thermodynamic and medicine, to materials sciences. An inelastic energy exchange permits to determinate the frequency of the molecular vibrations in an object. One can identify the molecules and thus, can characterize the object of study in a label-free way. Nevertheless, this method is afflicted with faults. Beside the presence of fluorecence that can drown the Raman answer, the main drawback is the long exposition time required. In the case of biological sample, this can prohibit the use of spontaneous Raman scattering for microspectroscopy measures: the spectral mapping of microscopic objects. To avoid this problem, new techniques have been developed. It is the case of Coherent anti-Stokes Raman scattering (CARS) spectroscopy. Due to its coherence and its directivity, the anti-Stokes signal has an intensity 105 to 106 times greater than the spontaneous Raman scattering one. The exposition time is then reduced to a tolerable level for biological objects during microspectroscopy measures. Moreover, the anti-Stokes characteristic of the signal prevents the fluorescence contribution. However, a major fault still limits the use of this technique: the nonresonant background. This phenomenon can diminish, even overwhelm the resonant contribution carrying the information. This thesis permitted the development of CARS approaches that allow the reduction of the nonresonant background. To do so, a multiplex CARS (M-CARS) spectroscopy apparatus in a forward configuration has been built. Its abilities are illustrated with spectral measures of mineral, vegetal and biological samples. Based on this system, it has been established an innovative method that can discriminate the resonant signal from the nonresonant one thanks to a static electric field. It has been also been demonstrated the development of a process that has allowed the first M-CARS microspectroscopy measure of a biological sample in a contrapropagative configuration. This setup limits the collect of the signal to the object of study, avoiding the acquisition of the resonant and resonant signals coming from the solvent, responsible for the major part of non resonant background during a CARS measure in a forward configuration.

Microscopy techniques have been developed and refined over multiple decades, but innovation around single photon modalities has slowed. The advancement of the utility of information acquired, and minimum resolution available is seemingly reaching an asymptote. The fusion of light microscopy and well-studied nonlinear processes has broken through this barrier and enabled the collection of vast amounts of additional information beyond the topographical information relayed by traditional microscopes. Through nonlinear imaging modalities, chemical information can also be extracted from tissue. Nonlinear microscopy also can beat the resolution limit caused by diffraction, and offers up three-dimensional capabilities. The power of nonlinear imaging has been demonstrated by countless research groups, solidifying it as a major player in biomedical imaging. The value of a nonlinear imaging system could be enhanced if a reduction in size would permit the insertion into bodily cavities, as has been demonstrated by linear imaging endoscopes. The miniaturization of single photon imaging devices has led to significant advancements in diagnostics and treatment in the medical field. Much more information can be extracted from a patient if the tissue can be imaged in vivo, a capability that traditional, bulky, table top microscopes cannot offer. The development of new technologies in optics has enabled the miniaturization of many critical components of standard microscopes. It is possible to combine nonlinear techniques with these miniaturized elements into a portable, hand held microscope that can be applied to various facets of the biomedical field. The research demonstrated in this thesis is based on the selection, testing and assembly of several miniaturized optical components for use as a nonlinear imaging device. This thesis is the first demonstration of a fibre delivered, microelectromechanical systems mirror with miniaturized optics housed in a portable, hand held package. Specifically, it is designed for coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excitation fluorescence imaging. Depending on the modality being exploited, different chemical information can be extracted from the sample being imaged. This miniaturized microscope can be applied to diagnostics and treatments of spinal cord diseases and injuries, atherosclerosis research, cancer tumour identification and a plethora of other biomedical applications. The device that will be revealed in the upcoming text is validated by demonstrating all designed-for nonlinear modalities, and later will be used to perform serialized imaging of myelin of a single specimen over time.

Neurological disorders, including spinal cord injury, peripheral nerve injury, traumatic brain injury, and neurodegenerative diseases, pose significant challenges in terms of diagnosis, treatment, and understanding the underlying pathophysiological processes. Label-free multiphoton microscopy techniques, such as coherent Raman scattering, two-photon excited autofluorescence, and second and third harmonic generation microscopy, have emerged as powerful tools for visualizing nervous tissue with high resolution and without the need for exogenous labels. Coherent Raman scattering processes as well as third harmonic generation enable label-free visualization of myelin sheaths, while their combination with two-photon excited autofluorescence and second harmonic generation allows for a more comprehensive tissue visualization. They have shown promise in assessing the efficacy of therapeutic interventions and may have future applications in clinical diagnostics. In addition to multiphoton microscopy, vibrational spectroscopy methods such as infrared and Raman spectroscopy offer insights into the molecular signatures of injured nervous tissues and hold potential as diagnostic markers. This review summarizes the application of these label-free optical techniques in preclinical models and illustrates their potential in the diagnosis and treatment of neurological disorders with a special focus on injury, degeneration, and regeneration. Furthermore, it addresses current advancements and challenges for bridging the gap between research findings and their practical applications in a clinical setting.

Les techniques de microscopie non linéaire connaissent un essor considérable ensciences du vivant, du fait de leur capacité à imager les tissus biologiques en profondeur et àexploiter différents contrastes dont les plus connus sont la fluorescence excitée à deux photons(2PEF) et la génération de second harmonique (SHG).Ce travail de thèse ’articule autour de la métrologie de milieux diffusants et d’applicationsbiomédicales de la microscopie non linéaire. Après une présentation générale de la technique, nousdécrivons une méthode originale de mesure du coefficient de diffusion μs et du facteur d’anisotropieg de milieux turbides épais basée sur la comparaison des intensités de fluorescence épi-collectéesselon trois modalités de notre microscope non-linéaire. Notre méthode est alors appliquée à lacaractérisation de gels biomimétiques et d’échantillons d’intérêt biologique. Le manuscrit abordeensuite le problème de l’imagerie en profondeur d’explants de peau humaine ré-innervée par desneurones sensoriels de rats nouveau-nés. Le choix du marqueur neuronal fluorescent fait l’objetd’une mesure in situ de la section efficace d’absorption à deux photons de différents fluorophores.La faisabilité d’une imagerie bimodale exploitant la fluorescence de ce marqueur et la réponse SHGdu collagène fibrillaire du derme est démontrée. Le manuscrit s’achève par une étude faisant suiteà des travaux de thèse antérieurs relatifs à la quantification de la fibrose hépatique par microscopieSHG/2PEF couplée. Nous appliquons la méthode de scoring SHG développée précédemment àune cohorte de patients infectés par le virus de l’hépatite C et comparons nos résultats aux testsMETAVIR et Ishak. / Nonlinear microscopy techniques are experiencing a considerable growth in lifescience, thanks to their ability to image biological tissues at high depth with different contrastssuch as two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG).This manuscript focuses on metrology of scattering media and on biomedical applications ofnonlinear microscopy. After an overview of the technique, we describe a novel method for measuringthe scattering coefficient μs and anisotropy factor g of thick turbid media based on the comparisonof fluorescence intensities epi-collected through the three modalities of our nonlinear microscope.Our method is then applied to biomimetic gels and to biological samples. The manuscript thentackles the problem of imaging deeply human skin explants re-innervated by sensory neurons fromneonatal rats. The choice of the fluorescence probe is the subject of an in situ measurement ofthe two-photon action cross-sections of various fluorophores. The feasibility of bimodal nonlinearimaging of re-innervated skin explants based on the 2PEF signal of the molecular probe and onthe SHG response of fibrillar collagen of the dermis is demonstrated. The manuscript ends witha study subsequent to the work of previous thesis regarding the quantification of liver fibrosisby SHG/2PEF microscopy. We apply the method of fibrillar collagen scoring by SHG previouslydeveloped to a new cohort of patients infected with hepatitis C virus and we analyze our resultsin terms of METAVIR and Ishak tests.

La microscopie non-linéaire résolue en polarisation est un outil puissant pour accéder à des informations structurelles dans les assemblages biomoléculaires. Les interactions non-linéaires entre matière et lumière induisent des processus complexes où des champs électromagnétiques cohérents interagissent avec les dipôles de transitions moléculaires. Le contrôle de la polarisation des champs électromagnétiques excitateurs et l’étude des réponses non-linéaires induites procurent de riches informations sur la distribution angulaire des molécules présentes dans le volume focal de l’objectif du microscope. Dans cette thèse, nous appliquons cette sensibilité à la polarisation à plusieurs modalités de microscopie cohérentes sans marquage (diffusion cohérente Raman anti-Stokes (CARS), diffusion Cohérente stimulée (SRS)) et à la fluorescence à deux photons (2PEF) afin d’obtenir des informations quantitatives sur la forme de la distribution moléculaire et l’orientation des lipides dans les membranes artificielles, ainsi que dans les membranes biologiques telles que la myéline des tissus de la moelle épinière. Avec cette technique, nous adressons une question fondamentale sur le comportement des ensembles lipidiques dans les membranes et sur l’effet d’autres molécules telles que le cholestérol et les marqueurs fluorescents. Nous démontrons que le CARS résolu en polarisation permet d’accéder à de fines informations sur l’organisation des lipides dans les membranes de la myéline, en deçà de la limite de diffraction. / Polarization resolved nonlinear microscopy is a powerful tool to image structural information in biomolecular assemblies. Nonlinear interaction between light and matter lead to complex processes where coherent combinations of optical fields couple to assemblies of molecular transition dipoles. Controlling polarized optical fields and monitoring nonlinear induced signals in a medium can nevertheless bring rich information on molecular orientational organization within the focal spot of a microscope objective. In this PhD thesis we apply this polarization sensitivity to different label-free optical coherent techniques (coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS)) and to two-photon fluorescence (2PEF) to retrieve quantitative information on the static molecular distribution shape and orientation of lipids in model membranes and biological membranes such as myelin sheaths in spinal cord tissues. With this technique, we address fundamental questions about lipid packing behavior in membranes, and how it can be affected by other molecules such as cholesterol and the insertion of fluorescent lipid probes. We demonstrate that polarization resolved CARS give access to fine details on lipids arrangement in myelin sheaths, at a sub-diffraction scale. In the context of experimental autoimmune encephalomyelitis disease (EAE) we show, that even at the stage of disruption of the myelin envelope during the demyelination process, lipids multilayers reveal strong capability to preserve their macroscopic self-assembly into highly organized structures, with a degree of disorganization occurring only at the molecular scale.

Nous avons développé une méthodologie basée sur phénomène de mélange à quatre ondre polarimétrique « Four wave Mixing FWM » et son équivalen résonant la diffusion Raman cohérente anti-Stokes (CARS, Coherent Anti-Stokes Raman Scattering) polarimétrique et réalisé des mesures sur des systèmes cristallins, simili biologiques : les membranes cellulaires connues sous le nom de « Multilamellar Vesicles MLV » et des échantillons de biologiques : la myeline, et ce, en variant les polarisations des lasers excitateurs, Pompe et Stokes. Le signal anti-Stokes émis est ensuite analysé afin d’en extraire les ordres 2 et 4 de la fonction de distribution angulaire des molécules actives constituant l’échantillon. Pour cela, plusieurs approches sont explorées telles que des algorithmes d’optimisation ou par décomposition en série de fourrier du signal polarimétrique. Ces multiples approches en traitement du signal permettent d’obtenir de manière rapide les coefficients des fonctions de distribution angulaire recherchées, et ainsi d’avoir des informations sur la symétrie des échantillons imagés, allant jusqu’à l’observation d’une symétrie d’ordre 4. La capacité de la microscopie non linéaire résolue en polarisation à sonder des ordres moléculaires est clairement démontrée et ainsi son intérêt dans l’étude de la relation entre la structure et la fonction de systèmes biologiques. / The capacity to quantify molecular orientational order in tissues is of a great interest since pathologies (skin lesion, neurodegenerative diseases, etc) can induce strong modifications in proteins’ organization. While numerous studies have been undertaken using polarization resolved second order nonlinear optical microscopy which is only specific to non-centrosymmetric organizations, higher order effects have been less explored. Four-wave mixing (FWM) microscopy and its resonant counterpart coherent anti-Stokes Raman scattering (CARS) can be of a great utility as label free diagnosis tools benefiting from less constraining symmetry rules. In this work, we implement incident polarizations tuning in FWM and CARS microscopy to probe molecular order, using a generic method to read-out symmetry information.Fourier analysis of the polarization-resolved FWM/CARS signal processed with an analytical model provides a fast and direct determination of the symmetry orders of the distribution function of the probed molecules. This method does not require a priori knowledge of the organization structure and provides quantitatively its second and fourth order symmetries. We applied this technique on different systems, from crystalline to less organized (multilamellar vesicles and proteins aggregates). We show that this new approach brings additional and more refined information on supra-molecular structures in complex media.