The nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut. / text

Fluorescência é uma técnica bastante utilizada para diagnóstico, análise de materiais e tecidos biológicos, técnicas forenses entre outras. Neste contexto, métodos para detectar sinais de moléculas fluorescentes com maior sensibilidade e especificidade têm sido investigados, principalmente para detecção de moléculas em concentrações extremamente baixas. Dada a importância do tema, o presente trabalho, de caráter inovador, busca gerar nanoestruturas em prata com laser pulsado femtossegundo, capazes de aumentar níveis de fluorescência de moléculas próximas às nanoestruturas. Foi utilizado um laser Libra femtossegundo de 450 mW, 1 KHz de frequência e comprimento de onda de 850 nm da Coherent para a criação de nanoestruturas em prata pura polida. Diferentes parâmetros de marcação resultaram em variados perfis de nanoestruturas, tanto periódicas e regulares, quanto aglomerados caóticos de esferas nanométricas pela superfície marcada, onde as estruturas apresentavam periodicidade de aproximadamente 500 nm e as esferas possuem tamanhos variando de 50 a 500 nm, quando avaliadas por Microscopia Eletrônica de Varredura (MEV) e Microscopia de força atômica (AFM). O efeito de proximidade destas nanoestruturas caóticas com a adição de um fluoróforo (Protoporfirina IX à 0,5 μg/ml em etanol) proporcionou um aumento do sinal de fluorescência, quando comparado à uma região não marcada, quando avaliado por microscopia confocal de fluorescêcia. Portanto, este aumento de sinal foi de aproximadamente 25 vezes para excitação de um fóton (405 nm) e cerca de 300 vezes para a excitação de dois fótons (800 nm). / Fluorescence is a widely applied technique in diagnosis, material and biological tissue analysis, forensic sciences and other areas. Tools for enhancing the fluorescence signal with high sensitivity and specificity are needed to detect trace levels of target molecules. This innovative project aims to create nanostructures on pure silver using femtosecond pulsed laser to enhance the fluorescence signal emission from molecules near that interface. It was used a femtosecond Libra laser of 450 mW, 1KHz of frequency and wavelength of 850 nm from Coherent to form the nanostructures on polished pure silver. The nanostructures were obtained on different shapes onto the surfaces, from periodic nanostructures having ~500 nm of periodicity, to chaotic agglomerates of silver spheres with size ranging from 50 to 500 nm, when analyzed with Scanning Electron Microscopy and Atomic Force Microscopy. The effect of proximity between the chaotic structures and the fluorophore (Protoporphyrin IX at 0,5 μg/ml in ethanol) resulted in an increase of 25 times the fluorescence signal when used one photon excitation (405 nm) and enhancement of 300 times using two photon excitation.

This paper reports the surface morphologies and ablation of crystalline silicon wafers irradiated by infra-red 775 nm Ti:sapphire femtosecond laser. The effects of energy fluences (below and above single-pulse modification) with different number of pulses were studied. New morphological features such as pits, cracks formation, Laser-Induced Periodic Surface Structures (LIPSS) and ablation were observed. The investigation indicated that there are two distinct mechanisms under femtosecond laser irradiation: low fluence regime with different morphological features and high fluence regime with high material removal and without complex morphological features. / Singapore-MIT Alliance (SMA)

Nonlinear optical microscopy (NLOM) offers many advantages when imaging intact biological samples. By using ultrafast lasers in the near infrared and two photon excitation (TPE), signal production is limited to the focal volume and provides an excellent means for rendering thin, microscopic images from within the sample. Exogenous fluorophores/lumiphores may be used as efficient contrast agents to tag specific targets and provide enhanced signal. The efficiency of the TPE process in these contrast agents is broadly assumed to vary inversely with the laser pulsewidth, τ. In this work, we investigate the TPE efficiency of transform limited broadband (~133nm, ~10fs) and narrowband (~11nm, ~170fs) pulses in the generation of twophoton luminescence from semiconductor nanocrystals or quantum dots (QD’s) both theoretically and experimentally. Compared to standard organic dyes, QD’s possess a relatively broad, uniform spectral response that enables better use of the full bandwidth from the broadband laser. Theoretical calculations including both degenerate and non-degenerate TPE indicate a rolloff from the 1/τ behavior as the pulses’ spectral bandwidth becomes broader than the absorption spectra of the QD’s. Experimentally measured enhancement in luminescence intensity while using a broadband pulse is compared with the simulated enhancement in two-photon luminescence. A combination of increased understanding of the excitation processes in NLOM and proper selection of contrast agents will help in advancing the role of broadband ultrafast lasers in NLOM.

I studied a family of closely connected topics related to the production and application of ultrashort laser pulses. I achieved broadband cascade Raman generation in crystals, producing mutually coherent frequency sidebands which can possibly be used to synthesize optical pulses as short as a fraction of a femtosecond (fs). Unlike generation using gases, there is no need for a cumbersome vacuum system when working with room temperature crystals. Our method, therefore, shows promise for a compact system. One problem for sideband generation in solids is phase matching, because the dispersion is significant. I solved this problem by using non-collinear geometry. I observed what to our knowledge is a record-large number of spectral sidebands generated in a popular Raman crystal PbWO4 covering infrared, visible, and ultraviolet spectral regions, when I applied two 50 fs laser pulses tuned close to the Raman resonance. Similar generation in diamond was also observed, which shows that the method is universal. When a third probe pulse is applied, a very interesting 2-D color array is generated in both crystals. As many as 40 anti-Stokes and 5 Stokes sidebands are generated when a pair of time-delayed linear chirped pulses are applied to the PbWO4 crystal. This shows that pulses with picosecond duration, which is on the order of the coherence decay time, is more effective for sidebands generation than Fourier transform limited fs pulses. I also studied the technique of fs coherent Raman anti-Stokes scattering (CARS) which is used as a tool for detecting dipicolinic acid, the marker molecule for bacterial spores. I observed that there is a maximum when the concentration dependence of the near-resonant CARS signal is measured. I presented a model to describe this behavior, and found an analytical solution that agrees with our experimental data. Theoretically, I explored a possible application for single-cycle pulses: laser induced nuclear fusion. I performed both classical and quantum mechanical calculations for a system of two nuclei moving under a superintense ultrashort field. From our calculation I noted that the nuclear collisions occur on a sub-attosecond time scale, and are predicted to result in an emission of zeptosecond bursts of light.

Nonlinear optical microscopy (NLOM) offers many advantages when imaging intact biological samples. By using ultrafast lasers in the near infrared and two photon excitation (TPE), signal production is limited to the focal volume and provides an excellent means for rendering thin, microscopic images from within the sample. Exogenous fluorophores/lumiphores may be used as efficient contrast agents to tag specific targets and provide enhanced signal. The efficiency of the TPE process in these contrast agents is broadly assumed to vary inversely with the laser pulsewidth, τ. In this work, we investigate the TPE efficiency of transform limited broadband (~133nm, ~10fs) and narrowband (~11nm, ~170fs) pulses in the generation of twophoton luminescence from semiconductor nanocrystals or quantum dots (QD's) both theoretically and experimentally. Compared to standard organic dyes, QD's possess a relatively broad, uniform spectral response that enables better use of the full bandwidth from the broadband laser. Theoretical calculations including both degenerate and non-degenerate TPE indicate a rolloff from the 1/τ behavior as the pulses' spectral bandwidth becomes broader than the absorption spectra of the QD's. Experimentally measured enhancement in luminescence intensity while using a broadband pulse is compared with the simulated enhancement in two-photon luminescence. A combination of increased understanding of the excitation processes in NLOM and proper selection of contrast agents will help in advancing the role of broadband ultrafast lasers in NLOM.

Femtosecond time scale allows us to follow and control atomic and molecular motion. The atomic vibrations happen in the range of femtosecond scale. Thus, femtosecond technology effectively measures the atomic vibration. However, to determine electron motion, one needs to reach sub-femtosecond time scale that is in attosecond time scale. High Harmonic Generation (HHG) is a non-linear process that converts infrared light to shortest wavelength, such as in the XUV regime. HHG allows to explore electronic motion and to control electron dynamics. HHG easily reaches to XUV region and is enabling attosecond pulse generation. In this thesis we focused to generate attosecond pulses by using noble gases and their mixtures. We used only argon gas, only hydrogen molecule and their mixture with neon gas. We wanted to improve the conversion efficiency (10^-6) of the fundamental light into high harmonics. We use Ne and H2 gas mixture to look enhancement of the HHs.

Doctor of Philosophy / Department of Physics / Artem Rudenko / Christopher M. Sorensen / The motion of electrons in atoms, molecules, and solids in the presence of intense electromagnetic radiation is an important research topic in physics and physical chemistry because of its fundamental nature and numerous practical applications, ranging from precise machining of materials to optical control of chemical reactions and light-driven electronic devices. Mechanisms of light-matter interactions critically depend on the dimensions of the irradiated system and evolve significantly from single atoms or molecules to the macroscopic bulk. Nanoparticles provide the link between these two extremes. In this thesis, I take advantage of this bridge to study light-matter interactions as a function of nanoparticle size, shape, and composition. I present here three discrete, but interconnected, experiments contributing to our knowledge of nanoparticle properties and their response to intense, short-pulsed light fields. First, I investigate how individual nanoparticles interact with each other in solution, studying their temperature-dependent solubility. The interaction potential between 5.5nm gold nanoparticles, ligated by an alkanethiol was found to be -0.165eV, in reasonable agreement with a phenomenological model. The other two experiments explore ultrafast dynamics driven by intense femtosecond lasers in isolated, gas-phase metallic and dielectric nanoparticles. Photoelectron momentum imaging is applied to study the response of gold, silica, and gold-shell/silica-core nanoparticles (ranging from single to several hundred nanometers in size) with near-infrared (NIR), 25 fs laser pulses in the intensity range of 10¹¹ - 10¹⁴ W/cm². These measurements, which constitute the bulk of my graduate work, reveal the complex interplay between the external optical field and the induced near-field of the nanoparticle, resulting in the emission of very energetic electrons that are much faster than those emitted from isolated atoms or molecules exposed to the same light pulses. The highest photoelectron energies (“cutoffs”) were measured as a function of laser intensity, nanoparticle material and size. We found that the energy cutoffs increase monotonically with laser intensity and nanoparticle size, except for the gold/silica hybrid where the plasmon resonance response modifies this behavior at low intensities. The measured photoelectron spectra for metallic nanoparticles display a large energy enhancement over silica. Finally, the last part of this thesis explores the possibility to apply time-resolved x-ray scattering as a probe of the ultrafast dynamics in isolated nanoparticles driven by very intense (~10¹⁵ W/cm²) NIR laser radiation. To do this, I developed and built a nanoparticle source capable of injecting single, gas-phase nanoparticles with a narrow size distribution into the laser focus. We used femtosecond x-ray pulses from an x-ray free electron laser (XFEL) to map the evolution of the laser-irradiated nanoparticle. The ultrafast dynamics were observed in the single-shot x-ray diffraction patterns measured as a function of delay between the NIR and x-ray pulses, which allows for femtosecond temporal and nanometer spatial resolution. We found that the intense IR laser pulse rapidly ionizes the nanoparticle, effectively turning it into a nanoplasma within less than a picosecond, and observed signatures of the nanoparticle surface softening on a few hundred-femtosecond time scale.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15725号 / 工博第3339号 / 新制||工||1505(附属図書館) / 28270 / 京都大学大学院工学研究科原子核工学専攻 / (主査)教授 伊藤 秋男, 教授 河合 潤, 准教授 松尾 二郎 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16866号 / 工博第3587号 / 新制||工||1542(附属図書館) / 29541 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 平尾 一之, 教授 田中 勝久, 教授 三浦 清貴 / 学位規則第4条第1項該当