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Single-cell metabolic analysis by stimulated Raman scattering cytometryHuang, Kai-Chih 29 January 2020 (has links)
Understanding cellular heterogeneity has been a challenge in biology. Current bio-analytical methods such as mass spectrometry or fluorescence-based detection would destruct the sample or perturb the functions of targeted molecules. In situ imaging of bio-molecules at single cell level resolves the phenotypes at metabolomics domain, which can address the challenges in studying cellular heterogeneity. Stimulated Raman scattering (SRS) microscopy provides a label-free approach to identify molecules based on the signature of molecular vibrations. However, there are several challenges to overcome in order to use SRS as a single-cell analysis platform with high throughput, high content, and high sensitivity. My thesis work aims to overcome above-mentioned difficulties. To fulfill the first unmet need of single cell metabolic analysis, we developed an SRS flow cytometry, and demonstrated the discrimination of particles at a throughput of up to 11,000 particles per second, which is a four orders of magnitude improvement compared to conventional spontaneous Raman flow cytometry. Next, we addressed the second unmet need of single-cell metabolic analysis through the development of SRS imaging cytometry. Using this platform, we studied the response of human pancreatic cancer to drug-induced and starvation-induced stress, and discovered lipid-facilitated protrusion as a metabolic biomarker for stress-resistant cancer cells. Lastly, to probe low-concentration bio-molecules using fingerprint Raman bands, we utilized pre-resonance enhancement to increase the SRS signal by two orders of magnitude, and demonstrated ultra-sensitive imaging of retinoids in cells. We demonstrated in situ imaging of retinoid level in cancer cells and during neuronal development. Collectively, these efforts demonstrate SRS cytometry as a high-throughput, high-content, and high-sensitivity single-cell analysis platform with broad applications. / 2022-01-28T00:00:00Z
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Advancements in time resolved spectroscopy and nonlinear microscopySemon, Bryan 08 December 2023 (has links) (PDF)
Non-linear optical processes such as coherent anti-Stokes Raman scattering and sum frequency generation offer a view into the chemical and biological interactions of molecules that is distinctly different from linear techniques like near infra-red and fluorescence. This insight comes at a cost: non-linear techniques are more sensitive to external perturbations of the system, increasing the noise and decreasing the repeatability of the data. We work here on both aspects of these non-linear techniques, taking advantage of their power to offer new imaging techniques as well as working to quantify and reduce the non-resonant noise inherent to the system. In pursuit of the first part, we look at formalin fixed paraffin embedded tissue samples. This is the most common form of tissue storage in the world. However, the paraffin renders them unavailable for spectroscopic study. We introduce a new technique, combination coherent anti-Stokes Raman scattering microscopy and sum frequency generation microscopy, to avoid the issue of paraffin signal contamination. This high resolution, widefield technique allows for the separate identification of paraffin and the tissue embedded within it. We show in this work the capability of this technique to enable high throughput automated detection of osteoporosis in mice. In pursuit of the second part, we demonstrate experimentally for the first time, deferred build up in coherent anti-Stokes Raman scattering. We show that coherent anti-Stokes Raman scattering signal is maximized when the probe pulse is delayed by an amount dependent on the probe width and the material itself. Non-resonant contamination, however, is maximized when the probe delay is zero, meaning that it is possible to decrease the non-resonant noise while increasing the desired signal. We also show that the dephasing time is inversely dependent on the probe width, so narrower probe pulses allow for further delayed probe pulses, which in turn decrease non-resonant noise more. We demonstrate this technique by looking at the effects of hydrogen bonding in pyridine-water complexes.
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Sources optiques fibrées pour applications biomédicales / Fiber-based light source for biomedical applicationsHage, Charles-Henri 23 January 2013 (has links)
Ce mémoire présente les travaux effectués sur le développement d'une source optique servant à des applications d'imagerie biomédicale en général et de diffusion Raman cohérente en particulier. En effet la diffusion de ces dernières est freinée par le verrou technologique que constitue la nécessité de deux impulsions synchronisées et décalées en longueur d’onde. La praticité et les possibilités de conversions de fréquences offertes par l’optique non-linéaire fibrée sont ainsi utilisées pour adresser ce verrou technologique. Tout d’abord, une source simplement réglable en longueur d’onde est générée par l’effet d’auto-décalage en fréquence optique d'un soliton par effet Raman. Une étude des principaux paramètres de fibre aboutit à des décalages de 320 à plus de 500 nm, permettant une imagerie des résonances d’intérêt (≈ 1000-4000 cm-1). Deux applications de ce décalage sont présentées. Ensuite, l’autre impulsion voit sa largeur spectrale réduite de 70 à 10 cm-1 par compression spectrale, qui consiste en un "regroupement non-linéaire de fréquences sans pertes", afin de bénéficier de la résolution spectrale nécessaire. Enfin, la source développée est validée par l’acquisition de spectres CARS de différents échantillons de référence, pour différentes résonances (850 à 1750 cm-1). Une extension de la source à d'autres types d'imagerie est proposée, ainsi qu'une architecture de source quasiment entièrement fibrée exploitant les principes développés au cours de cette thèse / This manuscript presents the work done concerning the development of a light source used for biomedical imaging and more particularly for coherent Raman scattering imaging. In fact an efficient broadcasting of these ones is hampered by the need of two synchronized and wavelength shifted pulses. As so, the handiness and frequency conversion capabilities of nonlinear fiber optics are used to circumvent this technological lock. First of all, an easy wavelength tunable source is set by the use of the self-shifting in optical frequency of a soliton. A study of the main fiber parameters lead to shifts of 320 to more than 500 nm which allows interesting molecular resonances imaging (≈ 1000-4000 cm-1). Two applications of this shift are also reported. Then, the second pulse sees its spectral width reduced from 70 to 10 cm-1 by spectral compression, which consists in a "loss-less frequency regrouping", in order to obtain a proper spectral resolution. Finally, the developed source is validated by acquiring CARS spectra of different reference solvents and for different resonances (850 to 1750 cm-1). An evolution of this source to allow other imaging techniques is proposed, as well as a quasi-all-fibered source exploiting the principles addressed during this thesis work
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Implementation of second-order correlation spectroscopy (SOCOS) via all- Gaussian coherent Stokes and anti-Stokes Raman scatteringNagpal, Supriya 30 April 2021 (has links)
Powerful spectroscopic techniques increasingly involve nonlinear processes that arise due to the convolution of more than one electric field - input laser pulse. Analyzing the output of optical processes like these demands the utilization of deterministic improvement tools. Three-color coherent Raman scattering represents a complex non-degenerate four wave mixing process that includes contributions from both resonant and non-resonant interaction of the three input fields to generate a signal. In order to quantify these contributions, effective differentiation of the non- resonant (background) from the resonant (coherent signal) is required. These contributions can be differentiated based on how the molecular vibrational modes are being excited by the input pulses. The work described here demonstrates the ability of second-order correlation spectroscopy, applied along with an all-Gaussian theoretical model to analyze three color coherent Raman scattering processes. It is shown to discriminate between resonant versus non-resonant four wave mixing processes successfully. A robust, femtosecond/picosecond coherent Raman spectroscope is used to observe how the resonant signal builds up in a finite amount of time for different specimens and how it is can be controlled by input laser pulse shaping. A closed-form solution obtained via an all-Gaussian approach provides confirmatory theoretical proof of the experimental results obtained. This technique is used to study hydrogen bonding, which is a vital molecular interaction for bio-molecular systems and yet lacks a profound understanding of its ways of forming complexes. Furthermore, a novel second-order one-dimensional correlation function is introduced that replicates the results of the diagonal sum of the traditional synchronous two- dimensional correlation function, thus reducing a two-dimensional analysis to one-dimension. Along with the first demonstration of these analyses for coherent Raman scattering, a generalized approach is described, which opens up research opportunities to investigate these optical processes' dependence on multiple controlling parameters.
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Nonlinear optical endoscopy with micro-structured photonic crystal fibers / Endoscopie non-linéaire avec fibres optiques micro-structuréesLombardini, Alberto 13 December 2016 (has links)
Dans cette thèse, nous proposons l'utilisation d'un nouveau type de fibre à cristal photonique, la fibre Kagomé à coeur creux, pour la livraison d'impulsions ultra-courtes en endoscopie non linéaire. Ces fibres permettent la livraison d'impulsions sans distorsion sur une large bande spectrale, avec un faible bruit de fond, grâce à la propagation dans le cœur creux. Nous avons résolu le problème de la résolution spatiale, à l'aide d'une microbille en silice, insérée dans le cœur de la fibre Kagomé. Nous avons développé un système d'imagerie compacte, qui utilise un tube piézo-électrique pour le balayage du faisceau, un système achromatiques de microlentilles et une fibre Kagomé double gaine, spécialement conçue pour l'endoscopie. Avec ce système, nous avons réussi à imager des tissus biologiques, à l'extrémité distale de la fibre (endoscopie), en utilisant des différentes techniques tels que TPEF, SHG et CARS, un résultat qui ne trouve pas d'égal dans la littérature actuelle. L'intégration dans une sonde portable (4,2 mm de diamètre) montre le potentiel de ce système pour de futures applications en endoscopie multimodale in-vivo. / In this thesis, we propose the use of a novel type of photonic crystal fiber, the Kagomé lattice hollow core fiber, for the delivery of ultra-short pulses in nonlinear endoscopy. These fibers allow undistorted pulse delivery, over a broad transmission window, with minimum background signal generated in the fiber, thanks to the propagation in a hollow-core. We solved the problem of spatial resolution, by means of a silica micro-bead inserted in the Kagomé fiber large core. We have developed a miniature imaging system, based on a piezo-electric tube scanner, an achromatic micro-lenses assembly and a specifically designed Kagomé double-clad fiber. With this system we were able to image biological tissues, in endoscope modality, activating different contrasts such as TPEF, SHG and CARS, at the distal end of the fiber, a result which finds no equal in current literature. The integration in a portable probe (4.2 mm in diameter) shows the potential of this system for future in-vivo multimodal endoscopy.
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