Coherent control of laser induced processes is based on the quantum interference among multiple excitation pathways. Progress in the field has been fueled by advances in pulse shaping techniques, allowing modulation of phase and amplitude across the bandwidth of ultra short pulses. This dissertation makes use of coherent control technique for the optimization of two-photon fluorescence (TPF) and its applications in selective excitation for biomedical imaging. Different physical processes, e.g. TPF, second harmonic generation (SHG) and their ratios (e.g. TPF/SHG) were optimized by using feedback control pulse shaping technique with an evolutionary algorithm. Various nonlinear effects, e.g. filamentation, intensity clamping and white light generation were studied using two-photon fluorescence and Z-scan technique with different dyes and biomarkers. Simultaneous measurements of different nonlinear effects were performed. Novel methods were proposed and implemented to obtain two-photon excitation characteristics in intensity-resolved manner. Understanding of these nonlinear effects can give new solution to the issues of spatial resolution and molecular contrast for cellular and tissue imaging.

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Articular cartilage has been imaged using the following multi-photon modalities: Second Harmonic Generation (SHG), Two-photon Fluorescence (TPF) and Coherent Anti-Stokes Raman Scattering (CARS). A simple epi detection microscope was constructed for SHG and TPF imaging in the early stages of this research. Later the imaging was transferred to a new microscope system which allowed simultaneous forwards and epi detection and combined CARS imaging with TPF and SHG. Multiphoton spectroscopic studies were conducted on both intact tissue samples and the major components of the extracellular matrix, in order to identify sources of TPF. Fluorescence was detected from type II collagen, elastin and samples of purified collagen and elastin crosslinks. Age related glycation crosslinks of collagen may be a significant source of TPF. No fluorescence was detected from proteoglycans. In intact, unfixed healthy articular cartilage the cells were observed via CARS, surrounded in their pericellular matrix which is characterised by an increase in TPF. The collagen of the extra cellular matrix showed up clearly in the SHG images. Diseased cartilage was also imaged revealing microscopic lesion at the articular surface in early osteoarthritis and highly fibrous collagen structures and cell clusters in more advanced degeneration. In young healthy cartilage a network of elastin fibres were found lying parallel to the articular surface in the most superficial 50μm of the tissue. Regional variations in these fibres were also investigated. The fibres appeared mainly long and straight suggesting that they may be under tension, further work is needed to identify whether they have a mechanical function. The polarization sensitivity of the SHG from collagen has been investigated for both cartilage and tendon. In the most superficial tissue these measurements can be used directly to determine the collagen fibre orientation. However at increasing depths the effects of biattenuation and birefringence must be considered. Healthy cartilage has a characteristic pattern of polarization sensitivity with depth and this changes at lesions indicating a disruption of the normal collagen architecture. The methods developed in this thesis demonstrate the use of non-linear microscopy to visualise the structure of the extracellular matrix and cells in intact unstained tissue. They should also be appropriate in many areas of cell and matrix biology.

Polarization-dependence provides a unique handle for extending the dimensionality of optical microscopy, with particular benefits in nonlinear optical imaging. Polarization-dependent second order nonlinear optical processes such as second harmonic generation (SHG) provide rich qualitative and quantitative information on local molecular orientation distribution. By bridging Mueller and Jones tensor, a theoretical framework was introduced to experimentally extend the application of polarization-dependent SHG microscopy measurements toward in vivo imaging, in which partial polarization or depolarization of the beam can complicate polarization analysis. In addition, polarization wavefront shaping was demonstrated to enable a new quantitative phase contrast imaging strategy for thin transparent samples. The axially-offset differential interference contrast microscopy (ADIC) was achieved as a combination of classic Zernike phase contrast and Nomarski differential interference contrast (DIC) methods. The fundamentally unique manner of this strategy also inspired rapid volumetric analysis in time dimension that is accessible for most existing microscopy systems. Finally, the dimensionality of high speed twophoton fluorescence imaging was extended to the spectral domain by spatial/spectral multiplexing, enabling beam scanning two photon fluorescence microscopy with 17 frames per second rate and over 2000 effective spectral data points.<br>

Dans cette étude, nous avons développé un composé dérivé du NADPH, nommé Nanoshutter (NS). NS a été conçu pour inhiber l'activité catalytique de la NOS, c'est à dire la synthèse de NO, en occupant la place du NADPH dans le domaine réductase du NOS. La voie de synthèse de NO chez les mammifère correspond à l'oxydation de la L-arginine catalysée par la NOS, qui se produit dans son domaine oxygénase. Basée sur des données de modélisation moléculaire, la structure de NS est composée deux sous-unités: (i) le motif nucléotidique de reconnaissance du NADPH a été retenu, permettant au composé NS un ciblage approprié du site de liaison au NADPH de la NOS, (ii) le motif nicotinamide de NADPH a été remplacé par un groupe stilbène lié à un groupement terminal accepteur d'électrons. De plus, ce fragment est caractérisé par une très bonne section efficace d'absorption à deux photons (130 GM à 840 nm). NS1, le composé prototype de la famille NS, contient un groupe terminal NO2 en tant que groupe accepteur d'électrons. La valeur de Kd (~ 4,2 µM) a été estimée dans des expériences de titrage sous excitation un- ou deux-photons, et suggère une bonne affinité de liaison de NS1 à la NOS. De façon inattendue, NS1 présente une bonne sélectivité, en terme de rendement quantique de fluorescence, pour les isoformes de NOS par rapport à d'autres protéines qui contiennent ou non un site de liaison NADPH. En outre, il a été montré que NS1 inhibait de façon compétitive NOS par rapport au NADPH. Dans les expériences d'imagerie de fluorescence réalisées sur des cellules endothéliales (HUVEC), NS1 a démontré une internalisation rapide et efficace, avec un signal de fluorescence mis en évidence principalement dans la région périnucléaire, accompagné d’un signal plus sporadique à la membrane plasmique. Cette observation est en parfait accord avec la colocalisation de NS1 et eNOS mesurée par immunomarquage, démontrant ainsi que NS1 cible eNOS dans les cellules endothéliales. La vasoconstriction NO-dépendante attendue dans les anneaux aortiques isolés de souris a été montrée, mais uniquement en présence de catalase qui convertit H2O2 en H2O et O2. En revanche, en l'absence de catalase, la vasorelaxation a plutôt été observée. Ce résultat indique que la NOS n’est très certainement pas l’unique cible de NS1 dans le système endothélial, et que d’autres cibles en rapport avec la modulation de ROS (Reactive Oxygen Species) sont impliquées. En accord avec ce résultat, NS1 provoque une réponse biphasique de la production de ROS dans les cellules HUVEC : Une phase d'augmentation est observée aux faibles concentrations de NS1 (en dessous de 2 µM), suivie d'une diminution (inhibition de ROS) pour des concentrations de NS1 plus élevées. En outre, NS1 inhibe la production de O2- dans les macrophages de souris et les productions de H2O2 et de O2- survenant dans des conditions de découplage de nNOS in vitro. Des explications possibles pour interpréter ces données sont: NS1 probablement inhibe la production de ROS, soit produites au niveau de la NADPH oxydase ou (et) au cours du découplage de la NOS. L'origine de la phase d’augmentation reste plus difficile à interpréter, mais pourrait correspondre au ciblage de la glucose-6-phosphate deshydrogénase. Enfin, NS1 exerce un effet anti -angiogénique sur les cellules endothéliales et empêche la prolifération de cellules du mélanome. En conclusion, NS1 rempli l'objectif principal de cibler et inhiber la NOS en ciblant plus particulièrement le domaine réductase - il est aussi caractérisé par des propriétés d’absorption et de fluorescence à deux photons intéressantes permettant des applications in vitro et in vivo. L’ensemble de ces caractéristiques présentent un profil intéressant pour de futures applications d’imagerie en temps réel et non-invasive, avec également un fort potentiel pour des applications cliniques liées aux maladies NO-dépendantes. / In this study, we introduced a NADPH derivative named as Nanoshutter (NS). NS was designed to inhibit the catalytic activity of NOS, i.e. synthesis of NO, by occupying the NADPH site in the reductase domain of NOS. In mammals, NO participates in extensive physiological/pathological processes in the cardiovascular, nervous and immune systems. The pathway of mammal NO synthesis is the oxidation of L-arginine catalyzed by NOS, which occurs in its oxygenase domain. The catalysis requires three co-substrates (L-arginine, NADPH, and O2) and five cofactors groups (FAD, FMN, calmodulin, BH4 and heme). Guided by molecular modeling, the structure of NS contains two conjugated subunits: (i) the nucleotide recognition motif of NADPH was retained in NS, allowing a proper targeting to the NADPH binding site of NOS- (ii) the nicotinamide moiety of NADPH is replaced by a stilbene moiety linked with a terminal electron acceptor group, preventing electron flow from the reductase to the oxygenase domain of NOS. Furthermore, this moiety is characterized by a large two-photon absorption cross-section (130 at 840 nm). NS1, the first compound of the NS family, contains a NO2 terminal group as an electron acceptor group. NS1 displayed distinct fluorescence properties in its free and NOS-bound states. The Kd value (around 4.2 µM) was estimated in titration experiments performed under one- or two-photon excitation conditions, suggesting an effective binding of NS1 to NOS with a good affinity. Surprisingly, in terms of fluorescence quantum yield, NS1 displayed a good selectivity to the NOS isoforms over other proteins which contain or not a NADPH binding site. Furthermore, NS1 was shown to competitively inhibit nNOS in a dose-dependent manner. In fluorescence imaging experiments with endothelial cells (HUVEC), NS1 displayed a rapid and efficient internalization, with highlighted fluorescence signal at the perinucleus region and sporadic signal at the plasma membrane. This observation was in accordance with the colocalization imaging between NS1 and eNOS as shown by immunostaining, showing that NS1 actually targets eNOS in endothelial cells. The expected NO-dependent vasoconstriction in isolated mouse aortic rings was only evidenced in the presence of catalase, which converts H2O2 into H2O and O2. By contrast, in the absence of catalase, a contradictory vasorelaxation was observed. This result indicates that NS1 may target more than NOS in endothelium system, which is (are) likely related to Reactive Oxygen Species (ROS) production. Accordingly, NS1 led to a biphasic response of ROS production in HUVEC cells: An increasing phase occurred at low NS1 concentration (below 2 µM) and followed by a decreasing phase (ROS inhibition) at higher NS1 concentrations. Furthermore, NS1 was shown to inhibit O2- production in mouse macrophages and H2O2 and O2- production in uncoupled nNOS in vitro. Altogether, the possible but not exclusive explanations for current data are: in addition to its inhibition effect on NO production, NS1 probably also inhibits the ROS production either produced by NADPH Oxidase or by electron leakage from uncoupled NOS, or a combination of both. The origin of the increasing phase remains more elusive but could correspond to the targeting of glucose-6-phosphate-dehydrogenase (G6PD). Additionally, NS1 displayed anti-angiogenesis effect on endothelial cells and prevented proliferation of melanoma. In conclusion, NS1 fulfilled the goal as a new NOS inhibitor targeting the reductase domain and displayed a unique two-photon property in vitro and in vivo, these features may provide a promising future for non-invasive real-time imaging, and to potential clinical applications in the NO-dependent diseases.

This thesis provides an extensive study of the first molecular hyperpolarizability b of charge-transfer chromophores using hyper-Rayleigh scattering (HRS). The charge-transfer chromophores used in present work involve the tricyanohydrofuran¡]TCF¡^group as an electron acceptor, and/or thiophene in the pi-electron bridge. TCF is a very strong electron acceptor and thiophene greatly lowers the resonance energy. Their presence significantly increases the beta value of the chromophore, therefore enhancing potentials in applications. In hyper-Rayleigh scattering experiments, the laser radiation with tunable wavelengths is used as an excitation source for measuring the frequency dependence of beta. The experiment shows beta exhibiting a significant dispersion in the two-photon resonance region. Using the linear absorption spectrum in coordination with theory, we show that it is possible to use Kramers-Kronig (K-K) transform to reproduce the experimental beta value in the two-photon resonance region. The K-K approach provides an extension to the conventional Oudar-Chemla equation, which is invalid in the spectral region in which two-photon resonance occurs. Using the new approach, it is shown that reliable values of intrinsic hyperpolarizabilities beta_zero of charge-transfer chromophores can be extracted. The coordination of beta_zero with molecular structure provides one with an insight for the origin of the enhancement of the first molecular hyperpolarizability of charge-transfer chromophores. This thesis examines the variation of beta_zero with molecular structure. The same technique is also applied to a dendrimer that has charge-transfer nonlinear optical chromophores incorporated in the dendritic structure. The measured frequency dependent hyperpolarizability of the dendrimer is compared with that calculated from the linear absorption spectrum by the KK transform technique. The intrinsic hyperpolarizability beta_zero of the dendrimer obtained is compared with that of the single chromophore having a structure similar to that incorporated in the dendrimer. The comparison shows that the 3D dendritic structure is effective in reducing the interaction between chromophores by providing sufficient space between them, hence avoiding the possibility of aggregation formation due to attractive interactions between chromophores. The topic of two-photon fluorescence (TPF), which is related to HRS, is also investigated. The intensity of TPF is generally proportional to the square of the incident excitation intensity. Careful measurements of the TPF intensity of a nonlinear optical chromophore in conjunction with required auxiliary parameters have been used as a technique for determining the two-photon absorption cross-section. The TPF intensity measurement carried out in this thesis uses a variety of intensities. At low intensity excitation, the TPF intensity follows the usual quadratic intensity law (QIL), whereas deviations from the QIL are observed at higher incident intensities. The observation of similar lineshape associated with one- and two-photon fluorescence spectra suggests a 3-level model for the description of TPF excited by the incident intensity at various strengths. It is shown that by fitting the observed TPF intensity to an equation developed from the three-level model, it is possible to deduce the two-photon absorption cross section of the nonlinear optical chromophore in solution. The new technique developed using the three-level model is tested on a Rhodamine B/Chloroform solution. The two-photon absorption cross-section obtained by using the new technique is found in agreement with that reported in the literature. Having demonstrated the suitability of the new technique, it is used to determine the two-photon absorption cross-section of a novel nonlinear optical chromophore. The two-photon absorption cross-section using the new technique is then compared with that obtained by the nonlinear transmittance method. The two results are in good agreement, indicating the applicability of the new technique. The new technique is more convenient than the conventional low excitation TPF method as it does not require various auxiliary parameters, some of them are difficult to obtain. The second harmonic generation (SHG) of a chromophore/polymer film which is optically poled by using a coherent superposition of a fundamental and its second harmonic beams. The growth rate of the SHG intensity is found to be proportional to the fourth power of the incident intensity of the fundamental beam, and the plateau intensity SHG is proportional to the square of the incident intensity. These observations are not in agreement with the published theory. While the reason for disagreement is yet to be clarified, the information obtained is useful for the development of nonlinear optical devices.

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.

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).