Aseptic loosening of artificial joints is caused by an osteolytic reaction to wear debris mediated by macrophages and other cells. Imaging these wear particles within cells can be a key process in understanding particle-cell interactions. However, the compounds used in surgical implants are not easily visualised as no tagging molecule can be added without altering the properties of the material. We were interested in using a label free optical technique known as coherent anti-Stokes Raman scattering spectroscopy (CARS) to image these particles in cells. In this thesis we studied how to use CARS to image physiologically relevant wear particles within cells. We characterised the responses from our CARS system and found them to be in good agreement to the Raman spectra we obtained for the same materials. We showed that the forward scattered CARS signal was consistently larger than the backwards scattered signal for the same size particles, and also generated a larger contrast, especially between sub-micron sized particles and the non-resonant background. Wear particles of polyethylene isolated from a pin-on-plate wear simulator were shown to be in a similar size range to those retrieved from revision tissue. When incubated in our model macrophage cells we were able to image areas of CARS signal that indicated the location of these particles in the cell. Furthermore, using multiple CARS images taken at different Raman resonances we were able to distinguish between three different polymeric compounds added to cells, showing the specificity of the technique. The inherent 3D sectioning capabilities of multiphoton microscopy were used to generate projected images of the cells and contents, as well as estimating the particle loads within cells. These results show that CARS could be an important tool in imaging intra-cellular polyethylene and characterising the interactions of wear particles with cells and the surrounding tissue.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第18611号 / エネ博第307号 / 新制||エネ||63(附属図書館) / 31511 / 京都大学大学院エネルギー科学研究科エネルギー応用科学専攻 / (主査)教授 大垣 英明, 教授 白井 康之, 教授 松田 一成 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

Made available in DSpace on 2016-01-26T18:56:04Z (GMT). No. of bitstreams: 1 Tamiris Garbiatti de Oliveira.pdf: 1606086 bytes, checksum: cf9e26811174e34243865dc9dfffec23 (MD5) Previous issue date: 2015-03-23 / The unlimited use of pesticides, besides contribute with to increase environmental pollution, is also influencing negatively the health of the living beings, including the people health. Many studies show the result these chemicals in the reproductive system of animals and even same at man, can cause infertility problem, which can originate, among other factors, the low sperm quality and quantity. This is study aimed at evaluating human sperm quality parameters and the chemical evaluation of the core of the sperm trough Raman Scaterring Spectroscopy, after in vitro exposure to different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine. The choice of the use of the these herbicides was because they are widely use throughout the world, especially in Brazil, highlighted the west Paulista region, used at cane sugar plantation, significant culture of the region. In this context, sperm samples were selected according to the normal testing parameters that evaluate sperm quality, such as liquefaction, viscosity, appearance, volume, pH, sperm, vitality, morphology and motility. Semen samples of 10 healthy donor were exposed to study herbicides varying the concentration and time. After the different exposures (0.05, 0.5, 1.0, 5.0 and 125 &#956;g/mL, 30 and 60 min), the analysis of motility and vitality were again evaluated. The Raman Scaterring spectra were obtained from samples control sperm and after exposure to the herbicide 2,4-D e atrazine at concentration of 125 &#956;g/mL and 60 min incubation. The progressive motility and total motility decreased from the concentration 0,5 &#956;g/mL at times 30 and 60 minutes while the percentage of sperm properties increased. The progressive motility and total motility decreased from the concentration 0.5 mg / mL at times 30 and 60 minutes while the percentage of sperm immotile increased. The vitality (viable sperm) decreased significantly (p <0.05) from the exposure concentration 0.5 &#956;g/mL to 2,4-D and from 1.0 &#956;g/mL atrazine, at times 30 and 60 minutes, showing that there is a functional change of trend of human sperm after exposure to these herbicides, and this change is intensified with the exposure time factor and concentration of herbicides. Through the Raman Scaterring technique allowed us to characterize normal human sperm and exposed to herbicides and verify the change in the molecular structure in the exposed sperm, evidenced by changes in relative intensities of the bands 785, 1095, 1255, 1336, 1374, 1486 and 1575 cm-1 treated with 2,4-D and a decrease at 1095 after exposure to atrazine. These results indicate the modification of nitrogenous bases and the phosphate group that make up DNA present in the nucleus of the sperm. / A utilização desenfreada de praguicidas, além de contribuir com o agravamento da poluição ambiental, também está influenciando de forma negativa a saúde dos seres vivos, inclusive a saúde das pessoas. Muitos estudos mostram a interferência desses produtos químicos no sistema reprodutor de animais e até mesmo do homem, podendo acarretar problemas de infertilidade, que podem ser originados, dentre vários fatores, pela baixa quantidade e qualidade espermática. Este estudo visa á avaliação de parâmetros de qualidade espermática humana e a avaliação química do núcleo do espermatozoide, por meio de Espectroscopia de Espalhamento Raman, após a exposição in vitro a diferentes concentrações de ácido 2,4-diclorofenoxiacético (2,4-D) e atrazina. Estes herbicidas são amplamente utilizados em todo o mundo, principalmente no Brasil, em destaque a região do Oeste Paulista, por serem empregados na cana-de-açúcar, cultura significativa da região. Neste contexto, foram selecionadas amostras de espermatozoides de acordo com os parâmetros normais de testes que avaliam a qualidade espermática, como: liquefação, viscosidade, aparência, volume, pH, concentração de espermatozoides, vitalidade, morfologia e motilidade. Amostras de sêmen de 10 doadores saudáveis foram expostas aos herbicidas de estudo, variando o tempo e a concentração. Após as diferentes exposições (0,05; 0,5; 1,0; 5,0 e 125 &#956;g/mL, 30 e 60 min), as análises de motilidade e vitalidade foram novamente avaliadas. Os espectros de Espalhamento Raman foram obtidos das amostras de espermatozoide controle e após a exposição aos herbicidas 2,4-D e atrazina na concentração de 125 µg/mL em 60 min de incubação. A motilidade progressiva e motilidade total diminuíram a partir da concentração 0,5 &#956;g/mL nos tempos 30 e 60 min enquanto que a porcentagem de espermatozoides imóveis aumentou. A vitalidade (espermatozoides viáveis) diminuiu significativamente (p<0,05) a partir da exposição da concentração 0,5 &#956;g/mL para o 2,4-D e a partir de 1,0 &#956;g/mL para a atrazina, nos tempos 30 e 60min, mostrando-se que há uma tendência de alteração funcional do espermatozoide humano após a exposição a estes herbicidas, e esta alteração é intensificada com o fator de tempo de exposição e concentração dos herbicidas testados. Por meio da técnica de Espalhamento Raman foi possível caracterizar o espermatozoide humano normal e os expostos aos herbicidas e verificar a alteração na estrutura molecular nos espermatozoides expostos, evidenciadas pelas alterações nas intensidades relativas das bandas 785, 1095, 1255, 1336, 1374, 1486 e 1575 cm-1 do tratado com 2,4-D, e uma diminuição na banda de 1095 após a exposição à atrazina. Estes resultados indicam a alteração das bases nitrogenadas e do grupo fosfato que compõem o DNA presente no núcleo do espermatozoide.

Made available in DSpace on 2016-07-18T17:46:21Z (GMT). No. of bitstreams: 1 Tamiris Garbiatti de Oliveira.pdf: 1606086 bytes, checksum: cf9e26811174e34243865dc9dfffec23 (MD5) Previous issue date: 2015-03-23 / The unlimited use of pesticides, besides contribute with to increase environmental pollution, is also influencing negatively the health of the living beings, including the people health. Many studies show the result these chemicals in the reproductive system of animals and even same at man, can cause infertility problem, which can originate, among other factors, the low sperm quality and quantity. This is study aimed at evaluating human sperm quality parameters and the chemical evaluation of the core of the sperm trough Raman Scaterring Spectroscopy, after in vitro exposure to different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine. The choice of the use of the these herbicides was because they are widely use throughout the world, especially in Brazil, highlighted the west Paulista region, used at cane sugar plantation, significant culture of the region. In this context, sperm samples were selected according to the normal testing parameters that evaluate sperm quality, such as liquefaction, viscosity, appearance, volume, pH, sperm, vitality, morphology and motility. Semen samples of 10 healthy donor were exposed to study herbicides varying the concentration and time. After the different exposures (0.05, 0.5, 1.0, 5.0 and 125 &#956;g/mL, 30 and 60 min), the analysis of motility and vitality were again evaluated. The Raman Scaterring spectra were obtained from samples control sperm and after exposure to the herbicide 2,4-D e atrazine at concentration of 125 &#956;g/mL and 60 min incubation. The progressive motility and total motility decreased from the concentration 0,5 &#956;g/mL at times 30 and 60 minutes while the percentage of sperm properties increased. The progressive motility and total motility decreased from the concentration 0.5 mg / mL at times 30 and 60 minutes while the percentage of sperm immotile increased. The vitality (viable sperm) decreased significantly (p <0.05) from the exposure concentration 0.5 &#956;g/mL to 2,4-D and from 1.0 &#956;g/mL atrazine, at times 30 and 60 minutes, showing that there is a functional change of trend of human sperm after exposure to these herbicides, and this change is intensified with the exposure time factor and concentration of herbicides. Through the Raman Scaterring technique allowed us to characterize normal human sperm and exposed to herbicides and verify the change in the molecular structure in the exposed sperm, evidenced by changes in relative intensities of the bands 785, 1095, 1255, 1336, 1374, 1486 and 1575 cm-1 treated with 2,4-D and a decrease at 1095 after exposure to atrazine. These results indicate the modification of nitrogenous bases and the phosphate group that make up DNA present in the nucleus of the sperm. / A utilização desenfreada de praguicidas, além de contribuir com o agravamento da poluição ambiental, também está influenciando de forma negativa a saúde dos seres vivos, inclusive a saúde das pessoas. Muitos estudos mostram a interferência desses produtos químicos no sistema reprodutor de animais e até mesmo do homem, podendo acarretar problemas de infertilidade, que podem ser originados, dentre vários fatores, pela baixa quantidade e qualidade espermática. Este estudo visa á avaliação de parâmetros de qualidade espermática humana e a avaliação química do núcleo do espermatozoide, por meio de Espectroscopia de Espalhamento Raman, após a exposição in vitro a diferentes concentrações de ácido 2,4-diclorofenoxiacético (2,4-D) e atrazina. Estes herbicidas são amplamente utilizados em todo o mundo, principalmente no Brasil, em destaque a região do Oeste Paulista, por serem empregados na cana-de-açúcar, cultura significativa da região. Neste contexto, foram selecionadas amostras de espermatozoides de acordo com os parâmetros normais de testes que avaliam a qualidade espermática, como: liquefação, viscosidade, aparência, volume, pH, concentração de espermatozoides, vitalidade, morfologia e motilidade. Amostras de sêmen de 10 doadores saudáveis foram expostas aos herbicidas de estudo, variando o tempo e a concentração. Após as diferentes exposições (0,05; 0,5; 1,0; 5,0 e 125 &#956;g/mL, 30 e 60 min), as análises de motilidade e vitalidade foram novamente avaliadas. Os espectros de Espalhamento Raman foram obtidos das amostras de espermatozoide controle e após a exposição aos herbicidas 2,4-D e atrazina na concentração de 125 µg/mL em 60 min de incubação. A motilidade progressiva e motilidade total diminuíram a partir da concentração 0,5 &#956;g/mL nos tempos 30 e 60 min enquanto que a porcentagem de espermatozoides imóveis aumentou. A vitalidade (espermatozoides viáveis) diminuiu significativamente (p<0,05) a partir da exposição da concentração 0,5 &#956;g/mL para o 2,4-D e a partir de 1,0 &#956;g/mL para a atrazina, nos tempos 30 e 60min, mostrando-se que há uma tendência de alteração funcional do espermatozoide humano após a exposição a estes herbicidas, e esta alteração é intensificada com o fator de tempo de exposição e concentração dos herbicidas testados. Por meio da técnica de Espalhamento Raman foi possível caracterizar o espermatozoide humano normal e os expostos aos herbicidas e verificar a alteração na estrutura molecular nos espermatozoides expostos, evidenciadas pelas alterações nas intensidades relativas das bandas 785, 1095, 1255, 1336, 1374, 1486 e 1575 cm-1 do tratado com 2,4-D, e uma diminuição na banda de 1095 após a exposição à atrazina. Estes resultados indicam a alteração das bases nitrogenadas e do grupo fosfato que compõem o DNA presente no núcleo do espermatozoide.

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The objective of this dissertation is to shed light on the physical properties of InN epilayers grown by High-Pressure Chemical Vapor Deposition (HPCVD) for optical device applications. Physical properties of HPCVD grown InN layers were investigated by X-ray diffraction, Raman scattering, infrared reflection spectroscopies, and atomic force microscopy. The dependencies of physical properties as well as surface morphologies of InN layers grown either directly on sapphire substrates or on GaN/sapphire templates on varied growth conditions were studied. The effect of crucial growth parameters such as growth pressure, V/III molar ratio, precursor pulse separation, substrate material, and mass transport along the flow direction on the optical and structural properties, as well as on the surface morphologies were investigated separately. At present, growth of high-quality InN material by conventional growth techniques is limited due to low dissociation temperature of InN (~600 ºC) and large difference in the partial pressures of TMI and NH3 precursors. In this research, HPCVD technique, in which ambient nitrogen is injected into reaction zone at super-atmospheric growth pressures, was utilized to suppress surface dissociation of InN at high temperatures. At high pressures, long-range and short-range orderings indicate that c-lattice constant is shorter and E2(high) mode frequency is higher than those obtained from low-pressure growth techniques, revealing that InN structure compressed either due to a hydrostatic pressure during the growth or thermal contraction during the annealing. Although the influence of varied growth parameters usually exhibit consistent correlation between long-range and short-range crystalline orderings, inconsistent correlation of these indicate inclination of InN anisotropy. InN layers, grown directly on α-sapphire substrates, exhibit InN (1 0 1) Bragg reflex. This might be due to a high c/a ratio of sapphire-grown InN epilayers compared to that of GaN/sapphire-grown InN epilayers. Optical analysis indicates that free carrier concentration, ne, in the range of 1–50 × 1018 cm–3 exhibits consistent tendency with longitudinal-optic phonon. However, for high ne values, electrostatic forces dominate over inter-atomic forces, and consistent tendency between ne and LO phonon disappears. Structural results reveal that growth temperature increases ~6.6 ºC/bar and V/III ratio affects indium migration and/or evaporation. The growth temperature and V/III ratio of InN thin films are optimized at ~850 ºC and 2400 molar ratio, respectively. Although high in-plane strain and c/a ratio values are obtained for sapphire-grown epilayers, FWHM values of long-range and short-range orderings and free carrier concentration value are still lower than those of GaN/sapphire-grown epilayers. Finally, vibrational and optical properties of chalcopyrite ZGP crystal on the (001), (110), and (10) crystalline planes were investigated by Raman scattering and infrared (IR) reflection spectroscopies. Raman scattering exhibits a nonlinear polarizability on the c-plane, and a linear polarizability on the a- and b-planes of ZGP crystal. Also, birefringence of ZGP crystal was calculated from the hydrostatic pressure difference between (110) and (10) crystalline planes for mid-frequency B2(LO) mode.

<p dir="ltr">The objective of this research is to advance the development and application of laser diagnostics in gas phase medium, which ranges from atmospheric non-reacting flows to turbulent reacting flows in high-pressure, high-temperature environments. Laser diagnostic techniques are powerful tools for non-intrusive and in-situ measurements of important chemical parameters, such as temperature, pressure, and species mole fractions, in harsh environments. These measurements significantly advance the knowledge across various research disciplines, such as combustion dynamics, chemical kinetics, and molecular spectroscopy. In this thesis, detailed theoretical models and experimental analysis are presented for three different techniques: 1. Chirped-probe-pulse femtosecond coherent anti-Stokes Raman scattering (CPP fs CARS); 2. Two-color polarization spectroscopy (TCPS); 3. Ultrafast-laser-absorption-spectroscopy (ULAS). The first chapter provides a brief survey of laser diagnostics, including both linear and nonlinear methods. The motivations behind the three studies covered in this dissertation are also discussed. </p><p dir="ltr">In the second chapter, single-shot CPP fs CARS thermometry is developed for the hydrogen molecule at 5 kHz. The results are divided into two parts. The first part concentrates on the development of H₂ CPP fs CARS thermometry for high-pressure and high-temperature conditions. The second part demonstrates the application of H₂ CPP fs CARS in a model rocket combustor at pressures up to 70 bar. In the first part, H₂ fs CARS thermometry was performed in Hencken burner flames up to 2300 K, as well as in a heated gas-cell at temperatures up to 1000 K. It was observed that the H₂ fs CARS spectra are highly sensitive to the pump and Stokes chirp. Chirp typically originates from optical components such as windows and polarizers. As a result, the pump delay is modeled to provide a shift to the Raman excitation efficiency curve. With the updated theoretical model, excellent agreement was found between the simulated and experimental spectra. The averaged error and precision are 2.8% and 2.3%, respectively. In addition, the spectral phase and pump delay determined from the experimental spectra closely align with the theoretical predictions. It is also found that pressure does not have significant effects on the H₂ fs CARS spectra up to 50 bar at 1000 K. The collision model provides excellent agreement with the experiment. This allows the use of low-pressure laser parameters for high-pressure thermometry measurements. In the second part, spatially resolved H₂ temperature was measured in a rocket chamber at pressures up to 70 bar. This is the first demonstration of fs CARS thermometry inside a high-pressure rocket combustor. These results highlight the potential of using H₂ CPP fs CARS thermometry to provide quantitative data in high-pressure experiments for the study of combustion dynamics and model validation efforts at application relevant operating conditions.</p><p dir="ltr">The third chapter presents the development of a TCPS system for the study of the NO (<i>A</i>²Σ⁺-<i>X</i>²Π) state-to-state collision dynamics with He, Ar, and N₂. Two sets of TCPS spectra for 1% NO, diluted in different buffer gases at 295 K and 1 atm, were obtained with the pump beam tuned to the R₁₁(11.5) and ^OP₁₂(1.5) transitions. The probe was scanned while the pump beam was tuned to the line center. Collision induced transitions were observed in the spectra as the probe scanned over transitions that were not coupled with the pump frequency. The strength and structure of the collision induced transitions in the TCPS spectra were compared between the three colliding partners. Theoretical TCPS spectra, calculated by solving the density matrix formulation of the time-dependent Schrödinger wave equation, were compared with the experimental spectra. A collision model based on the modified exponential-gap law was used to model the rotational level-to-rotational level collision dynamics. An unique aspect of this work is that the collisional transfer from an initial to a final Zeeman state was modeled based on the difference in the cosine of the rotational quantum number <i>J</i> projection angle with the z-axis for the two Zeeman states. Rotational energy transfer rates and Zeeman state collisional dynamics were varied to obtain good agreement between theory and experiment for the two different TCPS pump transitions and for the three different buffer gases. One key finding, in agreement with quasi-classical trajectory calculations, is that the spin-rotation changing transition rate in the <i>A</i>²Σ⁺ level of NO is almost zero for rotational quantum numbers ≥ 8. It was necessary to set this rate to near zero to obtain agreement with the TCPS spectra. </p><p dir="ltr">The fourth chapter presents the development and application of a broadband ULAS technique operating in the mid-infrared for simultaneous measurements of temperature, methane (CH₄), and propane (C₃H₈) mole fractions. Single-shot measurements targeting the C-H stretch fundamental vibration bands of CH₄ and C₃H₈ near 3.3 μm were acquired in both a heated gas cell up to ~650 K and laminar diffusion flames at 5 kHz. The average temperature error is 0.6%. The average species mole fraction error are 5.4% for CH₄, and 9.9% for C₃H₈. This demonstrates that ULAS is capable of providing high-fidelity hydrocarbon-based thermometry and simultaneous measurements of both large and small hydrocarbons in combustion gases. </p>

<p dir="ltr">In recent years, nanosecond repetitively pulsed (NRP) plasma discharges have garnered significant interest due to their rapid generation of reactive excited-state species, reactive radicals, and localized heat release within nanosecond (ns) timescale. To effectively harness these plasmas for altering system-level thermal and chemical behavior, a thorough understanding of their governing physics is crucial. This knowledge enables the development of predictive plasma kinetic models for tailoring NRP plasmas to specific applications. However, achieving this requires high-fidelity experimental data to validate models and deepen our understanding of fundamental plasma physics. Advancing experimental spectroscopy and laser diagnostics methods is essential for probing such temporally highly dynamic and optically complex nonequilibrium environments. This includes developing novel <i>test platforms</i>, conducting <i>fundamental research</i> to address existing knowledge gaps, and constructing custom <i>ultrafast laser architectures</i> for probing plasma properties. </p><p dir="ltr">The pioneering development of Streak-based <i>test platform</i> in the diagnostics field of nanosecond pulsed plasmas and its successful application towards inferring the underlying ultrafast spatio-temporal evolution of nanosecond pulsed plasma discharges with an unprecedented time-resolution as short as ~25 ps is presented for the first time. Spectrally filtered, 1D line-imaging of nanosecond pulsed plasma discharges in a single-shot, jitter-free, continuously sweeping manner is obtained, and differences in discharge dynamics of air and N2 plasma environments are studied. Successive <i>test platform</i> advancement includes spectrally resolved Streak-spectroscopy measurements of thermal regime-transition evolution from early-nonequilibrium to local-thermal-equilibrium (LTE) to attain time-resolved quantitative insights into N2(C) state rotational/vibrational nonequilibrium temperatures, electron temperature/density, and spectral lifetime dynamics. </p><p dir="ltr">Ultrafast laser-based progression includes detailed <i>fundamental</i> investigation of higher-order optical nonlinearity perturbations of fs-EFISH by considering of – self-phase modulation induced spectral characteristic of fs-EFISH signal, calibration mapping during-below-and-beyond optical breakdown regime, optical Kerr effect consequences, impact of femtosecond (fs) laser seeding on the noninvasiveness of fs-EFISH, and spectral emission characteristics of fs laser filaments. To infer N2(X) state nonequilibrium of NRP pulsed plasmas, two hybrid fs/ps ro-vibrational coherent anti-Stokes Raman scattering (CARS) <i>ultrafast laser architectures</i> are developed. First architecture, single-laser-solution, reduces system’s energy budget by ~3 mJ/pulse for generating narrowband (~21 ps), high-energy (~420 μJ/pulse), 532 nm probe pulses through incorporation of custom built visible fs optical parametric amplifier (OPA) coupled with an Nd:YAG power amplifier module. The second architecture, two-laser-solution, improves system’s robustness through the development of a 1 kHz, 532 nm, high-energy (~600 μJ/pulse), low-jitter (<1 ps), narrowband (~27 ps), master-oscillator-power-amplification (MOPA) based picosecond probe pulse laser time-synchronized with fs master-oscillator. Single-shot, hybrid fs/ps narrowband ro-vibrational CARS demonstration in a combusting flame up to temperatures of ~2400 K is demonstrated. Experimental ro-vibrational CARS investigation includes polarization based nonresonant background suppression and demonstration of preferential Raman coherence excitation shift, a temperature sensitivity enhancing strategy for vibrationally hot mediums like nanosecond pulsed plasmas. Lastly, an ultrafast pulse-friendly optically accessible vacuum cell is designed and fabricated for controlled experiments of NRP fs/ps CARS. Special care is taken to prevent self-focusing and spectral-temporal chirp of fs CARS beams while maintaining Gaussian focusing beam caustic.</p>

<p>Ultrafast laser-based diagnostic techniques are powerful tools for the detailed understanding of highly dynamic combustion chemistry and physics. The ultrashort pulses provide unprecedented temporal resolution along with high peak power for broad spectral range−ideal for nonlinear signal generation at high repetition rate−with applications including next-generation combustors for gas turbines, plasma-assisted combustion, hypersonic flows and rotating detonation engines. The current work focuses on advancing (i) femtosecond (fs) two-photon laser-induced fluorescence, and (ii) hybrid femtosecond/picosecond vibrational and rotational coherent anti-Stokes Raman scattering (fs/ps RCARS and VCARS) to higher pressures for the first time. </p><p>Quantitative single-laser-shot kHz-rate concentration measurements of key atomic (O-atom) and molecular (CO) species is presented using femtosecond two-photon laser-induced fluorescence (TP-LIF) for a range of equivalence ratios and pressures in diffusion flames. A multitude of signal-interfering sources and loss mechanisms−relevant to high-pressure fs TP-LIF applications−are also quantified up to 20 atm to ensure high accuracy. The pressure scaling of interferences take into account degradation, attenuation and wave-front distortion of the excitation laser pulse; collisional quenching and pressure dependent transition line-broadening and shifting; photolytic interferences; multi-photon ionization; stimulated emission; and radiation trapping. </p><p>Hybrid fs/ps VCARS of N₂ is reported for interference-free temperature measurement at 1300-2300 K in high-pressure, laminar diffusion flames up to 10 atm. A time asymmetric probe pulse allowed for detection of spectrally resolved CARS signals at probe delays as early as ~200-300 fs while being independent of collisions for the full range of pressures and temperatures. Limits of collisional independence, accuracy and precision of the measurement is explored at various probe-pulse delays, pressures and temperatures. </p><p> </p><p>Additionally, a novel all diode-pumped Nd:YAG amplifier design is presented for generation of time-synchronized ps-probe pulses for hybrid fs/ps RCARS of N₂. High-energy, nearly transform-limited, single-mode, chirp-free ps probe-pulses are generated at variable pulsewidths. The detailed architecture and characterization of the laser is presented. kHz-rate RCARS thermometry is presented up to 2400 K. Excellent spatial, spectral, and temporal beam quality allowed for fitting the theoretical spectra with a simple Gaussian model for the probe pulse with temperature accuracies of 1-2%. </p> <p><br></p>