Single-cell bio-analytical techniques play a pivotal role in contemporary biological and biomedical research. Among current high-throughput single-cell imaging methods, coherent Raman imaging offers both high bio-compatibility and high-throughput information-rich capabilities, offering insights into cellular composition, dynamics, and function. Coherent Raman imaging finds its value in diverse applications, ranging from live cell dynamic imaging, high-throughput drug screening, fast antimicrobial susceptibility testing, etc. In this thesis, I first present a deep learning algorithm to solve the inverse problem of getting a chemically labeled image from a single-shot femtosecond stimulated Raman scattering (SRS) image. This method allows high-speed, high-throughput tracking of lipid droplet dynamics and drug response in live cells. Second, I provide image-based single-cell analysis in an engineered Escherichia coli (E. coli) population, confirming the chemical composition and subcellular structure organization of individual engineered E. coli cells. Additionally, I unveil metabolon formation in engineered E. coli by high-speed spectroscopic SRS and two-photon fluorescence imaging. Lastly, I present stimulated Raman-activated cell ejection (S-RACE) by integrating high-throughput SRS imaging, in situ image decomposition, and high-precision laser-induced cell ejection. I demonstrate the automatic imaging-identification-sorting workflow in S-RACE and advance its compatibility with versatile samples ranging from polymer particles, single live bacteria/fungus, and tissue sections. Collectively, these efforts demonstrate the valuable capability of SRS in high-throughput single-cell imaging and sorting, opening opportunities for a wide range of biological and biomedical applications.

No description available.

In this thesis the lateral resolution limits of the Laser Induced Forward Transfer (LIFT) technique are being investigated. LIFT is a laser direct write process with micron and below resolution and is suitable for modifying, repairing and prototyping micro-devices. Single laser pulses with wavelength of 800 nm and duration of 130 fs from a Ti:Sapphire laser system were focused onto a transparent donor substrate coated with thin film to transfer the thin film material in the form of micro-disks through a small air gap onto an acceptor substrate. In this thesis, donor glass substrate coated with 80nm continuous Cr film and also Cr disks array patterned by photolithography or e-beam lithography were used as targets. The ablation threshold and transfer threshold were determined experimentally and compared to results from two-temperature model (TTM) simulations and reasonably agreement was obtained. For the continuous film target, the size of the LIFT disks depend on the laser fluences and the smallest sizes of around 700 nm were obtained near the transfer threshold. For the pre-patterned disks array targets, initially 1.3m Cr disks were fabricated on the donor substrates by photolithography. Small focused, larger defocused and large top-hat laser beams were used to transfer the pre-patterned Cr disks. The morphology of the transferred material and reliability of transfer were studied. It was found that the large top-hat beam gave the most reliable and high quality transfer results, resulting in mostly intact LIFT disks on the acceptor substrate. To push the resolution limit further, 500nm Cr disks fabricated on the donor substrate by e-beam lithography were used. The successful transfer of these 500 nm Cr disks gives a positive indication that LIFT can potentially be extended further to the nano-scale regime (usually defined as having sub-100 nm resolution).

Blister-Based Laser-Induced Forward-Transfer (BB LIFT) is a new method of particle transfer, capable of projecting complex and fragile particles into the gas phase. The technique uses a laser pulse to deform a metal or polymer film on a transparent substrate. The deformation of the film creates a blister which can mechanically desorb particles adhered to the surface. This thesis covers the study of the underlying mechanisms of blister formation in relation to laser pulse duration and film properties, whilst also advancing upon the technique by developing new methods for particle transfer of 0-dimensional point particles, 1-dimensional nanotubes, and 2-dimensional crystals. Study of the blister formation was carried out on uncoated 200-400 nm Titanium films, using 120 fs and 7 ns laser pulses. The blisters were studied by Atomic Force Microscopy and optical analysis. Furthermore a theoretical model for the blister formation using ns laser pulses was developed using a linear heat transfer model, showing a good agreement between experiment and theory. From this model mechanisms for blister formation under both of these pulse durations were developed. It was concluded that ns laser pulses heat the thin film causing it to undergo thermal expansion where the temperature and thermal expansion properties of the film define the blister. Femtosecond pulses form blisters due to confined ablation of the film at the interface of the transparent substrate and the film. The expanding gas forces the metal to stretch, where the deformation is dictated by the Young’s modulus of the material with the major factor being the thickness of the titanium film. The velocity distribution of the desorbed material was studied by means of mass spectroscopy. An ionising laser pulse was focused a known distance from the donor film. The ejected particles crossed the laser beam, and with a controlled delay of the time between the blister pulse and ionisation pulse the velocities could be determined for fullerenes (C60) and gold coated silicon nanoparticles (Auroshells). Utilising C60 as the desorbed material we could identify that for ns BB-LIFT the C60 was emitted at a velocity mostly dependent upon the heat expansion coefficient for the titanium film, resulting in a velocity approximating 100 ms-1 with a secondary emission of fullerenes due to evaporation from the hot surface. However, for fs BB-LIFT this evaporated emission was not present and the velocities could be adjusted from 7-70 ms-1 by varying the Ti film thickness from 360 nm to 210 nm respectively. These results are consistent with the mechanisms proposed earlier. The spread of the desorbed particle beam was also studied for nanosecond and femtosecond laser-induced blisters utilising auroshells and C60. This was accomplished by placing a receiver platform at a known distance in front of the donor film in order to collect the desorbed particles. The radial spread was then analysed indicating a flat deposit approximately the size of the initial blister with a 5 degree spread from that point. This indicates that the desorbed beam is highly directional. From this it could be ascertained that the blisters do not form from a single point position on the film but expand uniformly with the area of laser irradiation defining the growth point of the blister. A problem with many molecular beam techniques is that large fragile molecules or nanoparticles cannot be introduced to the gas phase without causing damage to the particles. Studies into the desorption of Auroshells (150 nm diameter), C60 (1 nm), PCBM (a fragile exohedral fullerene), carbon nanotubes (1x1000 nm), and 2D films (1x10000x10000 nm) showed that these materials were successfully transferred from the donor film to a receiver plate without causing damage to the particles. This was determined via Raman, NMR, AFM, and SEM measurements. Lastly a technique that allowed the growth of carbon nanotubes directly on the donor film utilising a a multi-layered substrate was developed, enabling the removal and deposition of the nanotubes, without exposing them to any chemical treatment.

La structuration de matériaux transparents (verre, eau, ...) irradiés par des impulsions lasers intenses brèves (nanoseconde) et ultra-brèves (femtoseconde) trouve de nombreuses applications dans les domaines de la biomédecine, des nano-optiques ou encore de l'endommagement d'optiques par des lasers de puissance. Dans un premier temps, nous avons modélisé le processus d'éjection qui se produit lors du transfert d'un matériau liquide (eau, hydrogel) lorsque la cible est irradiée par un laser nanoseconde. Le matériau est ici chauffé par conduction thermique via un ablateur métallique, et l'éjection est réalisée via un processus purement hydrodynamique. Si l'on considère maintenant cette même technique réalisée avec un laser femtoseconde, on peut envisager de transférer des volumes de matière nanométriques, ce qui correspond à la taille typique d'une molécule. En régime femtoseconde, les processus d'absorption de l'énergie sont différents, de même que les échelles de temps sur lesquels ils se produisent. Si l'impulsion laser est suffisamment focalisée dans le matériau, un plasma se forme dans la zone d'absorption, où la densité d'énergie atteinte est supérieure à l'énergie de liaison des matériaux considérés (eau, silice, mica). Un modèle d'ionisation et de chauffage des électrons a été mis en place et a été couplé à un code de propagation instantanée des ondes électromagnétiques. Trois applications ont été étudiées, correspondant à trois configurations laser/cible différentes : (1) un processus de formation de jets liquides et solides prometteur pour la réalisation de nano-optiques, (2) la formation de nanocavités à l'intérieur d'un volume de silice pouvant servir comme stockage d'information, (3) l'étude des seuils d'endommagement et d'ablation de la silice en surface. Dans chaque cas, la densité d'énergie absorbée a été utilisé comme donné initiale pour le code d'hydrodynamique CHIC qui a permis de suivre l'évolution du matériau après l'irradiation : ondes de compression, changements de phase, etc... La résolution couplée de la propagation du laser et de son absorption dans la matière nous a permis de mener une étude à la fois qualitative et quantitative de l'interaction. La comparaison avec les données expérimentales a aboutit à l'amélioration du modèle d'absorption et de chauffage. / Abstract

Parallèlement à l’impression jet d’encre et au bioplotting, l’impression d'éléments biologiques assistée par laser (Laser Assisted Bioprinting : LAB) qui utilise le transfert vers l’avant induit par laser (Laser Induced Forward Transfer : LIFT) a émergé comme une méthode alternative dans l’assemblage et la micro–structuration de biomatériaux et de cellules. Le LAB est une technique d’écriture directe qui offre la possibilité d’imprimer des motifs avec une haute résolution spatiale à partir d'une large gamme de matériaux solides ou liquides, tels que des diélectriques, des biomolécules et des cellules vivantes en solution.Dans nos travaux de recherche, nous avons considéré une approche expérimentale et numérique pour étudier les mécanismes physiques mis en jeu lors de la microimpression d’éléments biologiques assistée par laser. Dans un premier temps nous avons défini les paramètres rhéologiques des bioencres et les conditions de transfert (composition, épaisseur et viscosité de la bioencre et énergie laser). Puis nous avons mené une analyse statistique du volume des gouttelettes déposées pour quatre viscosités de bioencre, cinq épaisseurs de bioencre et cinq énergies laser. Ensuite nous avons conçu et mis en place un système d’imagerie résolue en temps pour étudier les effets de la viscosité sur la dynamique de l’éjection. Nous avons ainsi différencié trois régimes d'éjection en fonction de l'énergie laser déposée dans la couche absorbante, de la viscosité et de l'épaisseur de la bioencre. Parallèlement, un modèle numérique a été mis en place pour comprendre et prédire la dynamique de l’éjection en fonction de paramètres multiples : choix et épaisseur de la couche absorbante, épaisseur de la couche de bioencre, énergie laser déposée. Enfin, au regard de ces études, nous proposons un mécanisme d'éjection des microgouttelettes intervenant au cours du procédé de microimpression assistée par laser. / Over this decade, cell printing strategy has emerged as one of the promising approaches to organize cells in two and three dimensional engineered tissues. In parallel with ink-jet printing and bioplotting, Laser Assisted Bioprinting (LAB) using Laser-Induced Forward Transfer (LIFT) has emerged as an alternative method in the assembly and micropatterning of biomaterials and cells. LAB is a laser direct-write technique that offers the possibility of printing micropatterns with high spatial resolution from a wide range of solid or liquid materials, such as dielectrics, biomolecules and living cells in solution. In our research works, we considered an experimental and numerical approach to study the physical mechanisms involved in the biological elements microprinting laser assisted.First we defined the rheological parameters of bioinks and the transfer conditions (composition, thickness and viscosity of the bioink and laser energy). Then we led a statistical analysis of the volume of the transfer droplets for four viscosities of bioink, five thicknesses of bioink and five laser energies. Then we designed and implemented a system for time resolved imaging to study the effects of viscosity on the dynamics of the ejection. Thus we have differentiated three ejection regimes in function of the laser energy released in the absorbing layer, the visocsity and the thickness of the bioink. In parallel, a numerical model was developed to understand and predict the dynamics of the ejection parameters according to multiple choice and thickness of the absorbing layer, thickness of the layer bioencre, energy deposited. Finally, with regard to these studies, we propose a mechanism for ejecting droplets involved in the process of laser-assisted microprinting.

O poli (p-fenilenevinileno), ou PPV, é um polímero de grande relevância tecnológica devido a suas propriedades eletroluminescentes, que têm sido exploradas em diodos emissores de luz orgânicos, displays flexíveis e outros dispositivos optoeletrônicos. Embora o PPV seja um material de importância para muitas aplicações, a sua síntese na nano/microescala não pode ser obtida através do método padrão, o qual utiliza o aquecimento de um polímero precursor poli (cloreto de xileno tetrahidrotiofenio) (PTHT). Este trabalho mostra como a microestruturação com pulsos de femtosegundo pode ser empregada para a síntese de PPV em regiões pré-determinadas, empregando três diferentes abordagens, permitindo uma nova metodologia para a fabricação precisa de microcircuitos poliméricos complexos, (i) na primeira abordagem, o processo de conversão é obtido pela irradiação de filmes de PTHT com pulsos laser ultracurtos em regiões previamente determinadas, o que leva ao controle espacial da formação de PPV em microescala, (ii) na segunda abordagem, microestruturas tridimensionais dopadas com PTHT foram fotopolimerizadas por absorção de dois fótons. A conversão de PTHT para PPV nestas microestruturas dopadas foi obtida após um tratamento térmico, (iii) na terceira abordagem, a transferência direta induzida por laser (LIFT) com pulsos de femtossegundos permite a deposição controlada de PPV com alta resolução espacial, fornecendo micropadrões 2D, preservando sua estrutura e propriedades ópticas. As estruturas foram caracterizadas por microscopia eletrônica de varredura, microscopia óptica de transmissão, microscopia de fluorescência e microscopia confocal de fluorescência. Suas propriedades ópticas foram analisadas através de sistemas de micro-fotoluminescência e micro-absorção implementadas em um microscópio invertido. Medidas de espectroscopia Raman, microscopia de força atômica e medidas elétricas também foram realizadas. Este trabalho mostra como a microestruturação com laser de fs pode ser explorada para a síntese de PPV em regiões pré-determinadas para fabricar uma variedade de microdispositivos, abrindo novos caminhos na optoeletrônica baseada em polímeros. / Poly(p-phenylenevinylene), or PPV, is a polymer of great technological relevance due to its electroluminescent properties, which have been exploited in organic light emitting diodes, flexible displays and other optoelectronic devices. Although PPV is a material of foremost importance for many applications, its synthesis at the nano/micro scale cannot be achieved through the standard method that uses heating of a precursor polymer poly(xylene tetrahydrothiophenium chloride)(PTHT). This work demonstrates the use of direct laser writing with femtosecond pulses to obtain the synthesis of PPV in pre-determined regions, by applying three different approaches, allowing the precise fabrication of complex polymeric microcircuits, (i) in the first approach the conversion process is achieved by irradiating PTHT films with ultra-short laser pulses in previously determined regions, which leads to the spatial control of PPV formation at microscale, (ii) in the second approach, three-dimensional microstructures doped with PTHT were photopolymerized by two photons absorption. The conversion of PTHT to PPV in these doped microstructures was obtained by a subsequent thermal treatment, (iii) in the third approach, laser-induced forward transfer (LIFT) with femtosecond pulses enables the controlled deposition of PPV with high spatial resolution, providing 2D micropatterns, while preserving its structure and optical properties. The structures were characterized by scanning electron, fluorescence, transmission and confocal fluorescence microscopies. Their optical properties were analyzed by micro-photoluminescence and micro-absorption setups assembled on an inverted microscope. Raman spectroscopy, electrical measurements and atomic force microscopy were also performed. This thesis shows the use of fs-laser writing methods for the synthesis of PPV in pre-determined regions, to fabricate a variety of microdevices, thus opening new avenues in polymer-based optoelectronics.

O poli (p-fenilenevinileno), ou PPV, é um polímero de grande relevância tecnológica devido a suas propriedades eletroluminescentes, que têm sido exploradas em diodos emissores de luz orgânicos, displays flexíveis e outros dispositivos optoeletrônicos. Embora o PPV seja um material de importância para muitas aplicações, a sua síntese na nano/microescala não pode ser obtida através do método padrão, o qual utiliza o aquecimento de um polímero precursor poli (cloreto de xileno tetrahidrotiofenio) (PTHT). Este trabalho mostra como a microestruturação com pulsos de femtosegundo pode ser empregada para a síntese de PPV em regiões pré-determinadas, empregando três diferentes abordagens, permitindo uma nova metodologia para a fabricação precisa de microcircuitos poliméricos complexos, (i) na primeira abordagem, o processo de conversão é obtido pela irradiação de filmes de PTHT com pulsos laser ultracurtos em regiões previamente determinadas, o que leva ao controle espacial da formação de PPV em microescala, (ii) na segunda abordagem, microestruturas tridimensionais dopadas com PTHT foram fotopolimerizadas por absorção de dois fótons. A conversão de PTHT para PPV nestas microestruturas dopadas foi obtida após um tratamento térmico, (iii) na terceira abordagem, a transferência direta induzida por laser (LIFT) com pulsos de femtossegundos permite a deposição controlada de PPV com alta resolução espacial, fornecendo micropadrões 2D, preservando sua estrutura e propriedades ópticas. As estruturas foram caracterizadas por microscopia eletrônica de varredura, microscopia óptica de transmissão, microscopia de fluorescência e microscopia confocal de fluorescência. Suas propriedades ópticas foram analisadas através de sistemas de micro-fotoluminescência e micro-absorção implementadas em um microscópio invertido. Medidas de espectroscopia Raman, microscopia de força atômica e medidas elétricas também foram realizadas. Este trabalho mostra como a microestruturação com laser de fs pode ser explorada para a síntese de PPV em regiões pré-determinadas para fabricar uma variedade de microdispositivos, abrindo novos caminhos na optoeletrônica baseada em polímeros. / Poly(p-phenylenevinylene), or PPV, is a polymer of great technological relevance due to its electroluminescent properties, which have been exploited in organic light emitting diodes, flexible displays and other optoelectronic devices. Although PPV is a material of foremost importance for many applications, its synthesis at the nano/micro scale cannot be achieved through the standard method that uses heating of a precursor polymer poly(xylene tetrahydrothiophenium chloride)(PTHT). This work demonstrates the use of direct laser writing with femtosecond pulses to obtain the synthesis of PPV in pre-determined regions, by applying three different approaches, allowing the precise fabrication of complex polymeric microcircuits, (i) in the first approach the conversion process is achieved by irradiating PTHT films with ultra-short laser pulses in previously determined regions, which leads to the spatial control of PPV formation at microscale, (ii) in the second approach, three-dimensional microstructures doped with PTHT were photopolymerized by two photons absorption. The conversion of PTHT to PPV in these doped microstructures was obtained by a subsequent thermal treatment, (iii) in the third approach, laser-induced forward transfer (LIFT) with femtosecond pulses enables the controlled deposition of PPV with high spatial resolution, providing 2D micropatterns, while preserving its structure and optical properties. The structures were characterized by scanning electron, fluorescence, transmission and confocal fluorescence microscopies. Their optical properties were analyzed by micro-photoluminescence and micro-absorption setups assembled on an inverted microscope. Raman spectroscopy, electrical measurements and atomic force microscopy were also performed. This thesis shows the use of fs-laser writing methods for the synthesis of PPV in pre-determined regions, to fabricate a variety of microdevices, thus opening new avenues in polymer-based optoelectronics.

L'ingénierie tissulaire est reconnue comme une méthode potentielle pour réparer ou régénérer les tissus endommagés. Malgré de grandes avancées dans l'ingénierie tissulaire, la réussite de la construction de tissus complexes avec des réseaux vascularisés reste un défi. Dans les modèles d'angiogenèse actuels, les cellules endothéliales sont ensemencées au hasard, n'offrant pas de structure organisée. La technologie de bioimpression par laser offre une résolution d'impression précise. Par cette technique, les structures microvasculaires peuvent être construites pour la fabrication d'organes complexes, ou pour modéliser la progression de la maladie ou les modèles de réponse aux médicaments. Dans cette étude, des techniques de bio-impression au laser ont été utilisées pour étudier le guidage de l'angiogenèse in vitro. Deux techniques basées sur le laser, le transfert direct induit par laser (LIFT) et le transfert latéral induit par laser (LIST) sont utilisées. Comparée à LIFT, la technologie LIST offrait des conditions idéales pour l'impression cellulaire telles que la concentration cellulaire requise pour la formation du tubes endothéliaux et l'uniformité du motif désiré. Nous avons réalisé le modelage de la formation de structures de type capillaire dans des motifs organisés via l'impression LIST. Les constructions de type capillaire formées présentent des motifs uniformes. Les structures formées ont été analysées par microscopie confocale et reconstruction d'images 3D. Bien que le développement de la lumière endothéliale soit incomplet, la technique développée possède le potentiel d'atteindre une stabilisation et un développement de la lumière si l'on recrute un deuxième type de cellule tel que les fibroblastes ou les péricytes. / Tissue engineering has been well acknowledged as a potential method to repair or regenerate damaged tissues in the human body, fulfilling the limitations and shortage in autologous and organ transplantations. Despite great advances in engineering tissues with simple geometry and low requirement for oxygen and blood supply such as cartilage, skin and cornea, success in constructing 3D complex tissues with vascularized networks remains a major challenge. Angiogenesis plays an important role in vascular development in vivo. In current angiogenesis models, endothelial cells are seeded randomly not offering precise and desired patterning. Laser-based bioprinting technology offers precise and high cell printing resolution. By using laser-based bioprinting technology, microvascular structures can be constructed as a platform for complex organ fabrication, disease progression and drug response models. In this study, laser-based bioprinting techniques are employed to study angiogenesis guidance in vitro by patterning endothelial cells. Two laser-based techniques, Laser-Induced Forward Transfer (LIFT) and Laser-Induced Side Transfer (LIST) are used as patterning tools. Compared to LIFT, LIST technology provided ideal conditions for cell printing such as required cell concentration for endothelial tube formation and pattern uniformity. In this study, we achieved the guidance of capillary-like structure formation in desired patterns via LIST printing. The formed capillary-like constructs featured precise patterns and uniformity. The structures were analyzed by confocal microscopy, 3D image reconstruction and frozen section procedure. Though lumen development was incomplete, it possesses the potential to attain further stabilization and lumen development if recruiting a second cell type such as fibroblast or pericyte.