A compact multi-photon microscope (MPM) was designed and developed with the use of low-cost mode-locked fiber lasers operating at 1040nm and 1560nm. The MPM was assembled in-house and the system aberration was investigated using the optical design software: Zemax. A novel characterization methodology based on 'nonlinear knife-edge' technique was also introduced to measure the axial, lateral resolution, and the field curvature of the multi-photon microscope's image plane. The field curvature was then post-corrected using data processing in MATLAB. A customized laser scanning software based on LabVIEW was developed for data acquisition, image display and controlling peripheral electronics. Finally, different modalities of multi-photon excitation such as second- and third harmonic generation, two- and three-photon fluorescence were utilized to study a wide variety of samples from cancerous cells to 2D-layered materials.

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

Nonlinear optical microscopy (NLOM) utilizing femtosecond laser pulses is well suited for imaging living tissues. This work reports on the design and development of an optical fiber based multispectral NLOM developed around a laser generating broadband sub-10-fs pulses. An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within the NLOM. Mouse tail tendon was used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an effective method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Reconstruction of ultrashort pulses at the focal plane of the objective allows the excitation of multiple fluorescent probes simultaneously. Multiple fluorescent probe excitation and spectral discrimination is demonstrated using mixtures of fluorescent dye solutions and an in-vitro angiogenesis model containing human umbilical vein endothelial cells (HUVEC’s) expressing multiple fluorescent proteins. Sub-10-fs pulses can be propagated through polarization-maintaining single mode fiber (PMF) for use in NLOM. We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al2O3 oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. Nonlinear excitation of multiple fluorophores simultaneously and polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22342号 / 医博第4583号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 松田 秀一, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The use of receptor-targeted lipid microbubbles imaged by ultrasound is an innovative method of detecting and localizing disease. However, since ultrasound requires a medium between the transducer and the object being imaged, it is impractical to apply to an exposed surface in a surgical setting where sterile fields need be maintained. Additionally, the application of an ultrasound gel to the imaging surface may cause the bubbles to collapse. Multiphoton microscopy (MPM) is an emerging tool for accurate imaging of tissues and cells with high resolution and contrast. We have recently developed a novel method for detecting targeted microbubble adherence to the upregulated plectin-receptor on pancreatic tumor cells using MPM. Specifically, the third-harmonic generation response can be used to detect bound microbubbles to various cell types presenting MPM as an alternative and useful imaging method. This is an interesting technique that can potentially be translated as a diagnostic tool for the early detection of cancer and inflammatory disorders.

Les phénomènes de diffusion cohérente non-linéaire ont été récemment proposés en alternatives à la fluorescence comme processus de marquage en microscopie multiphotonique. Les matériaux couramment appliqués dans ce contexte buttent toutefois sur une limite inférieure en taille déterminée par le seuil de détection de signaux faibles en optique non-linéaire. Aucun des efforts récents en détection en génération de second-harmonique (GSH), qui est le processus non-linéaire d’ordre le plus bas, n’a permis de descendre à ce jour au-dessous d’une barrière en taille de 40nm même en ayant recours aux techniques de détection les plus sensibles telles que le comptage de photons uniques. Les nanoparticules (NPs) restent ainsi dans la famille des nano-diffuseurs de “grande“ taille. Il apparaît toutefois possible de déplacer de façon significative cette limite inférieure vers les plus petites tailles en substituant aux isolants diélectriques ou aux semi-conducteurs à grands gaps des particules quantiques (PQs) à base de semi-conducteurs à gaps directs.Dans ce travail, un nouveau type de nanosondes hautement non-linéaires a été conçu et développé de façon à franchir cette barrière de taille minimale pour atteindre l’échelle de nanoparticules uniques. Nous considérons ainsi l’excitation résonnante à deux photons de nanoparticules quantiques individuelles à base de CdTe (de la famille des “zinc-blendes”) d’un diamètre d’environ 12.5nm, qui fournissent une émission cohérente efficace par GSH jusqu’à hauteur de 105 comptages de photons par seconde. Elles présentent de plus l’avantage d’une remarquable sensibilité à l’orientation de leur réseau cristallin octupolaire.De plus, il a été démontré que les effets de confinement quantique déterminent fortement les caractéristiques de la susceptibilité non-linéaire du second-ordre χ(2). La caractérisation quantitative du χ(2) des PQs, en particulier leur dispersion spectrale et leur dépendance en taille est menée par spectroscopie de particules uniques ainsi qu’en moyenne d’ensemble par diffusion Hyper-Rayleigh (HRS). Nous fournissons en particulier la preuve que sous certaines conditions, le χ(2) de structures à base de semi-conducteurs en mode de confinement quantique peut très largement dépasser sa valeur en milieu massif. De plus, un nouveau type de PQs hybridant des semi-conducteurs en géométries de type “bâtonnet sur sphère” (BS) a été développé sur la base de composantes cristallines de symétries différentes, afin d’augmenter leur non-linéarité quadratique effective, tout en maintenant leur taille dans un régime proche d’un fort confinement quantique. Le nouveau tenseur hybride complexe χ(2) est analysé en terme d’interférence des susceptibilités constitutives, en prenant en compte les différentes formes et symétries associées aux composantes octupolaires et dipolaires.Il en résulte pour de telles structures une exaltation significative du χ(2), qui excède celle des PQs à constituant unique compte tenu du couplage entre matériaux non-linéaires et d’un temps de décohérence plus long, que nous attribuons à un effet de separation de charge photo-induit. / Nonlinear coherent scattering phenomena from single nanoparticles have been recently proposed as alternative processes for fluorescence in multiphoton microscopy staining. Commonly applied nanoscale materials, however, have reached a certain limit in size dependent detection efficiency of weak nonlinear optical signals. None of the recent efforts in detection of second-harmonic generation (SHG), the lowest order nonlinear process, have been able to cross a ~40 nm size barrier for nanoparticles (NPs), thus remaining at the level of “large” nanoscatterers, even when resorting to the most sensitive detection techniques such as single-photon counting technology. As we realize now, this size limitation can be significantly lowered when replacing dielectric insulators or wide gap semiconductors by direct-gap semiconducting quantum dots (QDs). Herein, a new type of highly nonlinear nanoprobes is engineered in order to surpass above mentioned size barrier at the single nanoparticle scale. We consider two-photon resonant excitation in individual zinc-blende CdTe QDs of about 12.5 nm diameter, which provide efficient coherent SHG radiation, as high as 105 Hz, furthermore exhibiting remarkable sensitivity to spatial orientation of their octupolar crystalline lattice. Moreover, quantum confinement effects have been found to strongly contribute to the second-order nonlinear optical susceptibility χ(2) features. Quantitative characterization of the χ(2) of QDs by way of their spectral dispersion and size dependence is therefore undertaken by single particle spectroscopy and ensemble Hyper-Rayleigh Scattering (HRS) studies. We prove that under appropriate conditions, χ(2) of quantum confined semiconducting structures can significantly exceed that of bulk. Furthermore, a novel type of semiconducting hybrid rod-on-dot (RD) QDs is developed by building up on crystalline moieties of different symmetries, in order to increase their effective quadratic nonlinearity while maintaining their size close to a strong quantum confinement regime. The new complex hybrid χ(2) tensor is analyzed by interfering the susceptibilities from each component, considering different shape and point group symmetries associated to octupolar and dipolar crystalline structures. Significant SHG enhancement is consequently observed, exceeding that of mono-compound QDs, due to a coupling between two nonlinear materials and slower decoherence, which we attribute to the induced spatial charge separation upon photoexcitation.