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La microscopie STED (stimulated emission depletion ou déplétion par émission stimulée) est une des méthodes les plus répandues de microscopie de super-résolution. Dans un microscope STED, un faisceau en anneau se superpose avec le faisceau d'excitation et éteint les fluorophores en périphérie du faisceau d'excitation par émission stimulée. Au centre de l'anneau, où le faisceau STED a une intensité nulle, la fluorescence reste intacte. Cette technique nécessite un montage complexe dans lequel deux faisceaux laser, en général issus de deux sources différentes, doivent être parfaitement alignés et superposés. Dans ce travail de thèse, nous proposons deux configurations STED qui ont pour but de simplifier le montage et de réduire le coût total d'un tel système. L'idée de base dans les deux cas est d'utiliser la même source laser à la fois pour l'excitation et la déplétion par émission stimulée. Dans la première configuration, nous avons développé une source bicolore originale basée sur un laser Nd-YAG microchip. Ce laser microchip délivre simultanément des impulsions sub- ns à deux longueurs d'onde, 355 nm (excitation) et 532 nm (déplétion), qui sont générés par conversion harmonique à partir d'une émission laser Nd-YAG et offrent l'avantage d'être intrinsèquement alignées et synchronisées. Afin de trouver des colorants appropriés pour cette source particulière, nous avons développé une méthode de caractérisation et testé différents colorants Nous avons construit un microscope à partir de cette source laser et obtenu des images avec une résolution améliorée. La réduction du volume d'excitation a été confirmée par spectroscopie de corrélation de fluorescence (FCS). Cependant, les aberrations chromatiques des optiques utilisées limitent les performances du montage actuel. Une perspective prometteuse serait de combiner le STED à la microscopie à feuille de lumière (SPIM), plus tolérante des défauts d'achromatisme, et nous montrons les premiers résultats de cette approche. Dans la seconde configuration, les aberrations chromatiques ne sont plus un problème puisqu' une seule longueur d'onde est utilisée pour l'excitation (par absorption à deux photons) et la déplétion. En jouant sur la durée de l'impulsion (et donc la valeur de l'intensité crête), un de ces deux procédés peut être favorisé. La fluorescence est excitée à deux photons par une impulsion femtoseconde, puis est éteinte par émission stimulée à un photon avec une impulsion étirée. Nous avons utilisé une technique résolue en temps (Time-Correlated Single Photon Counting - TCSPC) pour étudier l'efficacité de déplétion du colorant DCM en solution. Les simulations numériques montrent que cette méthode peut être appliquée à la microscopie de super résolution. En fin de cette partie, nous présentons les premières images obtenues avec un microscope développé au laboratoire qui permet l'excitation à deux photons et la déplétion à un photon avec une seule longueur d'onde, ainsi que l' amélioration de la résolution observée. Dans ce travail, nous avons donc mis en place expérimentalement, pour la première fois, deux concepts destinés à simplifier en utilisant deux sources laser originales. / Stimulated emission depletion (STED) is a well-known super-resolution method. In a STED microscope, a doughnut-shaped beam is superimposed with the excitation beam and keeps the fluorophores in the periphery of the excitation spot in a dark state by stimulated emission, thus effectively improving the spatial resolution in a scanning configuration. This technique requires a complex setup since two laser beams, generally from different sources need to be perfectly aligned. In this work we propose two STED configurations that will simplify the setup and reduce the total cost of such a system. The basic idea in both cases is to use the same laser source for both excitation and stimulated emission depletion. In the first setup we have developed an original two-color source based on a microchip Nd-YAG laser. This microchip laser simultaneously delivers sub-ns pulses at two wavelengths, 355 nm (excitation) and 532 nm (depletion), which are generated by harmonic conversion from an Nd-YAG laser emission and offer the advantage of being intrinsically aligned and synchronized. Further work consisted in determining suitable dyes for this particular source. We have built a microscope setup based on this laser source and obtained images with an improved resolution. The confirmation of the reduction of the excitation volume is showed by Fluorescence Correlation Spectroscopy (FCS) measurements. However, the performance of this system is limited by chromatic aberrations. The combination of Selective Plane Illumination Microscopy (SPIM) with STED is considered. In the second setup the chromatic aberrations are no longer a problem since the same wavelength is used for two photon excitation and one photon depletion. By playing on the duration of the pulse (thus the instantaneous intensity), one of these two processes can be favored. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. We used the Time Correlated Single Photon Counting (TCSPC) method to study the depletion efficiency of DCM dye in solution and numerical simulations show that this method can be applied to super-resolved microscopy. In the end we present the preliminary images obtained with a home-built Two-photon Single wavelength STED microscope and the resolution improvement obtained. Further improvements are to be made to the custom microscope. In this work we have experimentally implemented, for the first time, two concepts meant to simplify the STED setups by using original sources.

Cette thèse porte sur la description de l'assemblage des virus dans le cadre de la physique statistique ainsi que sur les méthodes de mesure de cet assemblage utilisant les marqueurs fluorescents. Nous nous y attachons à décrire la dynamique de l'agrégation des protéines aux échelles de la population et du virus unique. Nous proposons deux méthodes pour mesurer les grandeurs physiques associées : taille et forme de la structure finale d'une part, taux d'agrégation au cours de la croissance d'autre part. Dans ce travail, nous nous sommes intéressés à la description physique de l'auto-assemblage des protéines virales. La physique de l'auto-assemblage in-vitro des virus sphériques, dont la structure est déterminée par l'agencement régulier de leurs constituants protéiques, a été théoriquement et expérimentalement caractérisée auparavant par des modèles d'agrégation. Les modèles existants décrivaient l'assemblage à quantité de composants viraux fixée dans un système ferme à partir des constituants élémentaires du virus. In-vivo, la situation est bien entendu différente. Abstraction faite de la grande complexité du milieu cellulaire, les virus s'échappent de la cellule une fois formés pour aller infecter de nouvelles cellules. De plus, la quantité de constituants est sans cesse modifiée par la fabrication ou la dégradation des protéines virales. Enfin les méthodes de mesures utilisées in-vitro ne sont généralement plus envisageables in-vivo. Nous avons donc étudié les effets d'un flux de matière dans système ouvert via le calcul de l'état stationnaire, et via la résolution numérique des équations d'évolution des populations d'agrégats qui décrivent la cinétique d'agrégation des protéines virales. Dans ce cadre, nous avons mis en valeur le lien entre la description de l'état général du système en termes de populations et le devenir individuel d'un virus en formation pour le suivi duquel des méthodes expérimentales existent. Nous nous sommes alors attachés à proposer un traitement approprié de telles données expérimentales pour déterminer les valeurs des paramètres physiques du modèle / In this thesis work, we study the self-assembly of viral particles and focus on the analysis of measurements based on fluorescence labeling of viral proteins. We propose a theoretical model of the dynamic of viral proteins self-assembly at the cell membrane based on previous models developed to describe the in-vitro assembly of spherical viruses. We study the evolution of the populations in the successive stages of viral budding as well as the evolution of single particle within this framework. We also provide various data analysis to measure the physical values involved in the process: rate of aggregation during the bud growth, size and shape of the eventual structure. Viruses are biological objects unable to replicate without infecting an host cell since they lack part of the molecular machinery mandatory for genetic code replication and proteins production. Originally aimed at controlling the diseases they cause, the study of viruses is now rich of applications in medical and technological field (gene therapy, phage therapy, targeted therapy, bio-templating, cargo specific encapsulation, etc.). The existent models describing the self-assembly of viral proteins have successfully captured many features observed in the in-vitro experiments. We study the expected evolution when an open system is considered with an input flux of proteins and an output flux of released virion, characteristic of the in-vivo situation. We derive the population distribution at steady state and numerically study their dynamic under constant viral protein input flux. We also study the case of a single bud evolution which can be followed by its fluorescence emission. We study the possibility to estimate shape parameters at the single viral particle level such as radius and completion for the human immunodeficiency virus (HIV) from single molecule localization superresolution microscopy. These techniques known as (f)PALM or (d)STORM, record labeled proteins position with a precision of few to tens of nanometers. We propose an approach base on the maximum likelihood statistical method which is tested on both real and simulated images of fully formed particles. Our results suggest that it can offer a precision on the determination of the global structure finner than the positioning precision of the single proteins. This efficiency is however tempered when the parameter of interest does not affect the figures of merit to which the method is sensitive such as the apparent area and the image contours

Táto práce se zabývá použitím hlubokého učení neuronových sítí ke zvýšení rozlišení obrázků, které obsahují obličeje. Tato metoda najde uplatnění v různých oblastech, zejména v bezpečnosti, například, při bezpečnostním incidentu, kdy policie potřebuje identifikovat podezřelého z nahraného videa ze sledovací kamery. Cílem této práce je navrhnout minimálně dvě architektury neuronových sítí, které budou pracovat se sekvencí snímků, a porovnat je s metodami zpracování jediného snímku. Pro tento účel je také vytvořena nová trénovací množina, obsahující sekvenci snímku obličeje. Metody zpracování jednoho snímku jsou natrénované na nové množině. Dále jsou navrženy nové metody zvětšení obrázků na základě sekvence snímků. Tyto metody jsou založené na U-Net modelu, který je úspěšný v segmentaci, ale také v superrozlišení. Pro zlepšení architektury byly použity reziduální bloky a jejich modifikace, a navíc také percepční ztrátová funkce, která dovoluje vyhnout se rozmazání a získání více detailů. První čast této práce je věnovana popisu neuronových sítí a některých architektur, jejichž modifikace mohou být použity v superrozlišení. Druhá část se poté zabývá popisem metod pro zvýšení rozlišení obrazu pomocí jednoho snímku, několika snímků a videa. Ve třetí části jsou popsány navržené metody a experimenty a v poslední části porovnaná metod založených na jednom snímku a několika snímcích. Navržené metody jsou schopny získat více detailů v obraze, ale mohou produkovat artefakty. Ty lze ale poté eliminovat pomocí filtru, například Gaussova. Nové metody méně selhávají při detekci obličejů, a to je podstatné u identifikace člověka v případě incidentu.

In this effort a new measurement technique for the lateral Goos-Hanchen shift is developed, analyzed, and demonstrated. The new technique uses classical image formation methods fused with modern detection and analysis methods to achieve higher levels of sensitivity than obtained with prior practice. Central to the effort is a new mathematical model of the dispersion seen at a step shadow when the Goos-Hanchen effect occurs near critical angle for total internal reflection. Image processing techniques are applied to measure the intensity distribution transfer function of a new divergence model of the Goos-Hanchen phenomena providing verification of the model. This effort includes mathematical modeling techniques, analytical derivations of governing equations, numerical verification of models and sensitivities, optical design of apparatus, image processing

<p>Microscale environments provide significant resolution and distortion challenges with respect to measurement techniques; however, with improvements to existing techniques, it is possible to gather relevant data to better understand the thermal and fluidic mechanisms at such small scales in evaporating droplets.</p> <p> </p> <p>Infrared thermography provides several unique challenges at small scales. A primary issue is that the low native resolution of traditional infrared cameras significantly hamper the collection of details of microscale features. Furthermore, surfaces exhibiting vastly different emissivities, results in inaccurate temperature measurements that can only be corrected with irradiance-based emissivity maps of the surface; however, due to the resolution limitations of infrared thermography, these emissivity maps can also display significant errors. These issues are overcome through the use of multi-frame super-resolution. The enhanced resolution allows for better capture of microscale features, therefore, enhancing the emissivity map. A quantitative error analysis of the system is conducted to quantify the feature size resolution improvement as well as the smoothing effect of super-resolution reconstruction. Furthermore, a sensitivity analysis is conducted to quantify the impact of registration uncertainty on the accuracy of the reconstruction. Finally, the improved emissivity map from super-resolution is demonstrated to show the increased accuracy over low-resolution mapping.</p> <p> </p> <p>When applied to water droplets, particularly on nonwetting surfaces, infrared thermography is confounded by the presence of nonuniform reflectivities due to the spherical curvature of the liquid-air interface. Thus, when measuring the temperature along the vertical axis of a water droplet, it is necessary to correct the reflection. Using a controlled background environment, in conjunction with the Fresnel equations, it is possible to correct the reflective effects on the interface and calculate the actual temperature profile. This allows for a better understanding of the governing mechanisms that determine the thermal transport within the droplet. While thermal conduction is the primary transport mechanism along the vertical axis of the droplet, it is determined that the temperature drop is partially dampened by the convective transport from the ambient air to the liquid interface. From this understanding revealed by the measurements, the vapor-diffusion-based model for evaporation was enhanced to better predict evaporation rates.</p> <p> </p> <p>Further exploration into the mechanisms behind droplet evaporation on nonwetting surfaces requires accurate knowledge of the internal flow behavior. In addition, the influence of the working fluid can have a significant impact on the governing mechanisms driving the flow and the magnitude of the flowrate. While water droplet evaporation has been shown to be governed by buoyancy-driven convection on nonwetting substrates, similar studies on organic liquid droplets are lacking. Particle image velocimetry is effective at generating a velocity flow field, but droplets introduce distortion due to the refraction from the spherical interface of the droplet. As such, velocity correction using a ray-tracing approach was conducted to correct the velocity magnitudes and direction. With the velocity measurements, the flow was determined to be surface-tension-driven and showed speeds that are an order of magnitude higher than those seen in buoyancy-driven flow in water droplets. This resulted in the discovery that advection plays a significant role in the transport within the droplet. As such, the vapor-diffusion-governed evaporation model was adjusted to show a dramatic improvement at predicting the temperature gradient along the vertical axis of the droplet.</p> <p> </p> <p>Armed with the knowledge of flow behavior inside droplets, it is expected that droplets with aqueous solutions should exhibit buoyancy-driven convection. The final part of this work, therefore, leverages this phenomenon to enhance mixing during reactions. Colorimetry is a technique that is widely utilized to measure the concentration of a desired sample within some liquid; the sample reacts with a reagent dye the color change is measured, usually through absorbance measurements. In particular, the Bradford assay is used to measure protein concentration by reacting the protein to a Coomassie^TM Brilliant Blue G-250. The absorbance of the dye increases, most significantly at the 590 nm wavelength, allowing for precise quantitation of the amount of protein in the solution. A droplet-based reaction chamber with buoyancy-enhanced mixing has the potential to speed up the measurement process by removing the need for a separate pre-mixing step. Furthermore, the reduced volume makes the process more efficient in terms of reactant usage. Experimental results of premixed solutions of protein sample and reagent dye show that the absorbance measurement through a droplet tracks strongly with the protein concentration. When the protein sample and dye reagent are mixed <i>in situ</i>, the complex interaction between the reactants, the mixing, and the adsorption of protein onto the substrate creates a unique temporal evolution in the measured absorbance of the droplet. The characteristic peaks and valleys of this evolution track strongly with concentration and provide the framework for measurement of concentration in a droplet-based system.</p> <p> </p> <p>This thesis extends knowledge about droplet thermal and fluidic behavior through enhanced measurement techniques. This knowledge is then leveraged in a novel application to create a simple, buoyancy-driven colorimetric reaction setup. Overall, this study contributes to the field of miniaturized, efficient reaction and measurement devices.</p>

CoRoT (Convection, Rotation and planetary Transits) é uma missão espacial liderada pela Agência Espacial Francesa (CNES) em associação com diversos parceiros internacionais, entre eles o Brasil. Seus objetivos principais são o estudo da sismologia estelar e a procura por planetas extra-solares (exoplanetas). Ambos os programas científicos baseiam-se em uma fotometria de altíssima precisão e requerem observações ininterruptas de longa duração, possíveis somente a partir do espaço. Uma revisão da literatura indica a existência de três técnicas principais para a realização de fotometria estelar a partir de imagens capturadas em CCDs: fotometria por máscara (por abertura), por ajuste de limiar (por threshold), e por ajuste da resposta impulsiva do sistema de aquisição de imagens. A fotometria por máscara, ou por abertura, apresenta maior precisão para o registro de estrelas brilhantes, em cenários de maior estabilidade de atitude do satélite (situação de baixo jitter), e é a solução adotada a bordo pelo satélite CoRoT, por ser um algoritmo determinístico. A fotometria por ajuste da resposta impulsiva, também chamada de função de espalhamento do ponto (point-spread function, PSF), por sua vez, por levar em conta a resposta do sistema a uma fonte pontual de luz, permite a restauração da imagem original através de processos de deconvolução; apresenta maior precisão para estrelas fracas, ou em um cenário degradado, com perturbações devidas a radiação externa (stray light), ou em que o jitter de atitude do satélite seja elevado. Tal robustez é uma característica desejada no processo de restauração de imagens. Já a fotometria por threshold é aplicada somente em casos de jitter muito elevado e pobre conhecimento da resposta impulsiva do sistema, de modo que não é utilizada no satélite CoRoT. Este trabalho visa consolidar e potencializar a participação brasileira no projeto CoRoT e contribuir com os esforços associados à redução de dados da missão, através de uma proposta inovadora de fotometria híbrida, que se utilizará dos conhecimentos da PSF modelada do instrumento e da alta relação sinal/ruído alcançada com a fotometria por máscara realizada a bordo, baseada e fundamentada em resultados preliminares que atestam o potencial desta metodologia. Tal algoritmo permite um ganho substancial de precisão fotométrica em relação à técnica de abertura, resultando em uma melhor exploração dos dados disponíveis, dada a sua robustez em caso de degradação dos dados de entrada. / CoRoT (Convection, Rotation and planetary Transits) is a high precision photometry experience dedicated to stellar seismology and the search for extrasolar planets. The mission is led by the French Space Agency (CNES) in association with several French laboratories and international partners in Austria, Belgium, Germany, Spain and Brazil. Both scientific programs require great instrumental stability and long, uninterrupted observation runs, which take place simultaneously on adjacent regions of the sky. An overview of the literature displays three main techniques to perform stellar photometry from space CCD images: aperture photometry, threshold photometry and PSF-fitting photometry. Aperture photometry defines a mask which represents the CCD pixels to be summed up in the computation of the collected photon flux for a given star. This method presents very high precision for isolated, bright stars and for stable satellite attitude (low jitter scenarios). It is the data reduction technique implemented on-board, due both to link capacity constraints (given the large number of targets simultaneously observed by the instrument) and to its deterministic algorithm. Fitting photometry allows restoration of degraded stellar images through deconvolution processes, using the point spread function (PSF) of the optical instrument itself. This technique presents better performance for crowded fields and for faint stars; it also presents robustness in the presence of disturbances such as stray light or high satellite attitude jitter. Finally, threshold photometry takes into account only those pixels whose values are above a given pre-computed level. This method is used only in scenarios with excessive satellite depointing due to attitude jitter, or in cases where the instrumental PSF is poorly known. Therefore, it is not used in the CoRoT mission. This work intends to consolidate and strengthen the Brazilian share in the CoRoT Project, contributing to the efforts associated to the ground-based reduction of scientific data, through an innovative, hybrid photometry technique, which will take advantage of a high-resolution instrumental PSF and of the high signal-to-noise-ratio obtained in the on-board aperture photometry. Studies here described show that this methodology, compared to the Aperture technique, achieves gains in photometric precision and in the operational duty cycle, enabling robust and accurate data exploitation.

CoRoT (Convection, Rotation and planetary Transits) é uma missão espacial liderada pela Agência Espacial Francesa (CNES) em associação com diversos parceiros internacionais, entre eles o Brasil. Seus objetivos principais são o estudo da sismologia estelar e a procura por planetas extra-solares (exoplanetas). Ambos os programas científicos baseiam-se em uma fotometria de altíssima precisão e requerem observações ininterruptas de longa duração, possíveis somente a partir do espaço. Uma revisão da literatura indica a existência de três técnicas principais para a realização de fotometria estelar a partir de imagens capturadas em CCDs: fotometria por máscara (por abertura), por ajuste de limiar (por threshold), e por ajuste da resposta impulsiva do sistema de aquisição de imagens. A fotometria por máscara, ou por abertura, apresenta maior precisão para o registro de estrelas brilhantes, em cenários de maior estabilidade de atitude do satélite (situação de baixo jitter), e é a solução adotada a bordo pelo satélite CoRoT, por ser um algoritmo determinístico. A fotometria por ajuste da resposta impulsiva, também chamada de função de espalhamento do ponto (point-spread function, PSF), por sua vez, por levar em conta a resposta do sistema a uma fonte pontual de luz, permite a restauração da imagem original através de processos de deconvolução; apresenta maior precisão para estrelas fracas, ou em um cenário degradado, com perturbações devidas a radiação externa (stray light), ou em que o jitter de atitude do satélite seja elevado. Tal robustez é uma característica desejada no processo de restauração de imagens. Já a fotometria por threshold é aplicada somente em casos de jitter muito elevado e pobre conhecimento da resposta impulsiva do sistema, de modo que não é utilizada no satélite CoRoT. Este trabalho visa consolidar e potencializar a participação brasileira no projeto CoRoT e contribuir com os esforços associados à redução de dados da missão, através de uma proposta inovadora de fotometria híbrida, que se utilizará dos conhecimentos da PSF modelada do instrumento e da alta relação sinal/ruído alcançada com a fotometria por máscara realizada a bordo, baseada e fundamentada em resultados preliminares que atestam o potencial desta metodologia. Tal algoritmo permite um ganho substancial de precisão fotométrica em relação à técnica de abertura, resultando em uma melhor exploração dos dados disponíveis, dada a sua robustez em caso de degradação dos dados de entrada. / CoRoT (Convection, Rotation and planetary Transits) is a high precision photometry experience dedicated to stellar seismology and the search for extrasolar planets. The mission is led by the French Space Agency (CNES) in association with several French laboratories and international partners in Austria, Belgium, Germany, Spain and Brazil. Both scientific programs require great instrumental stability and long, uninterrupted observation runs, which take place simultaneously on adjacent regions of the sky. An overview of the literature displays three main techniques to perform stellar photometry from space CCD images: aperture photometry, threshold photometry and PSF-fitting photometry. Aperture photometry defines a mask which represents the CCD pixels to be summed up in the computation of the collected photon flux for a given star. This method presents very high precision for isolated, bright stars and for stable satellite attitude (low jitter scenarios). It is the data reduction technique implemented on-board, due both to link capacity constraints (given the large number of targets simultaneously observed by the instrument) and to its deterministic algorithm. Fitting photometry allows restoration of degraded stellar images through deconvolution processes, using the point spread function (PSF) of the optical instrument itself. This technique presents better performance for crowded fields and for faint stars; it also presents robustness in the presence of disturbances such as stray light or high satellite attitude jitter. Finally, threshold photometry takes into account only those pixels whose values are above a given pre-computed level. This method is used only in scenarios with excessive satellite depointing due to attitude jitter, or in cases where the instrumental PSF is poorly known. Therefore, it is not used in the CoRoT mission. This work intends to consolidate and strengthen the Brazilian share in the CoRoT Project, contributing to the efforts associated to the ground-based reduction of scientific data, through an innovative, hybrid photometry technique, which will take advantage of a high-resolution instrumental PSF and of the high signal-to-noise-ratio obtained in the on-board aperture photometry. Studies here described show that this methodology, compared to the Aperture technique, achieves gains in photometric precision and in the operational duty cycle, enabling robust and accurate data exploitation.

Imaging at the nanoscale and/or at remote locations holds great promise for studies in fields as disparate as the life sciences and materials sciences. One such microscopy technique, stimulated emission depletion (STED) microscopy, is one of several fluorescence based imaging techniques that offers resolution beyond the diffraction-limit. All current implementations of STED microscopy, however, involve the use of free-space beam shaping devices to achieve the Gaussian- and donut-shaped Orbital Angular Momentum (OAM) carrying beams at the desired colors –-- a challenging prospect from the standpoint of device assembly and mechanical stability during operation. A fiber-based solution could address these engineering challenges, and perhaps more interestingly, it may facilitate endoscopic implementation of in vivo STED imaging, a prospect that has thus far not been realized because optical fibers were previously considered to be incapable of transmitting the OAM beams that are necessary for STED. In this thesis, we investigate fiber-based STED systems to enable endoscopic nanoscale imaging. We discuss the design and characteristics of a novel class of fibers supporting and stably propagating Gaussian and OAM modes. Optimization of the design parameters leads to stable excitation and depletion beams propagating in the same fiber in the visible spectral range, for the first time, with high efficiency (>99%) and mode purity (>98%). Using the fabricated vortex fiber, we demonstrate an all-fiber STED system with modes that are tolerant to perturbations, and we obtain naturally self-aligned PSFs for the excitation and depletion beams. Initial experiments of STED imaging using our device yields a 4-fold improvement in lateral resolution compared to confocal imaging. In an experiment in parallel, we show the means of using q-plates as free-space mode converters that yield alignment tolerant STED microscopy systems at wavelengths covering the entire visible spectrum, and hence dyes of interest in such imaging schematics. Our study indicates that the vortex fiber is capable of providing an all-fiber platform for STED systems, and for other imaging systems where the exploitation of spatio-spectral beam shaping is required.