Light matter interactions have led to a great part of our current understanding of the universe. When light interacts with matter it affects the properties of both the light and the matter. Visible light, being in the region that the human eye can "see," was one of the first natural phenomenon we used to learn about our universe. The application of fundamental physics research has spilled over into other fields that were traditionally separated from physics, being considered two different sciences. Current physics research has applications in all scientific fields. By taking a more physical approach to problems in fields such as chemistry and biology, we have furthered our knowledge of both. Nanocrystals have many interesting optical properties. Furthermore, the size and properties of nanocrystals has given them applications in materials ranging from solar cells to sunscreens. By understanding and controlling their interactions with systems we can utilize them to increase our knowledge in other fields of science, such as biology. Nanocrystals exhibit optical properties superior to currently used fluorescent dyes. By replacing molecular dyes with nanoparticles we can reduce toxicity, increase resolution and have better cellular targeting abilities. They have also shown to have toxicity to cancer and antibacterial properties. With the understanding of how to target specific cells in vitro as well as in vivo, nanoparticles have the potential to be used as highly cell specific nanodrugs that can aid in the fight against cancer and the more recent fight against antibiotic resistant bacteria. This dissertation includes our work on bioimaging as well as our novel drug delivery system. An explanation of toxicity associated with ZnO nanoparticles and how we can use it and the nonlinear optical properties of ZnO for nanodrugs and nanoprobes is presented.

Laser scanning confocal microscopy (LSCM) is an optical imaging technique that provides improved resolution and sensitivity over conventional methods of optical microscopy. However, the cost of most commercial LSCM systems exceeds the financial limitations of many smaller laboratories. The design of a custom LSCM created at a fraction of the cost of a commercial model is discussed in this paper. The increase in the incidence rate of skin cancer in the world today is alarming, as such, it is essential to provide an early, rapid and effective method for in vivo diagnostics of human skin tissue. LSCM is capable of detecting alterations in skin morphology and configuration, as well as providing chemical composition information which may be indicative of the development of skin cancer. If developed successfully, LSCM could replace the current invasive biopsy procedures performed today with a quick, non-invasive optical scanning method that would prove beneficial for both patients and physicians alike.

Fluorescence microscopy has been a valuable tool in the field of biological science as it allows one to study the structure and interaction of protein complexes and organelles in living cells. However, conventional optical microscopy technique has been limited by a trade-off between spatiotemporal resolution, signal contrast, and photodamage to the biological samples. It means that an increase in spatial resolution or signal contrast comes at the cost of higher laser power, serial-scanning, or longer image acquisition time. Unfortunately, this leads to severe photobleaching and photodamage to the samples and/or limited throughput of imaging, which is highly challenging to be circumvented through only optical imaging technique. Therefore, one has turned to artificial intelligence (AI) in image processing, applying deep learning algorithms to different imaging modalities to overcome these traditional limitations in optical microscopy systems. Herein we present multiple strategies on how deep learning can be applied to solve challenging and fundamental problems in different fluorescence microscopy modalities. To do so, we present UNet-RCAN, a two-step deep learning network architecture based on a residual U-Net and residual channel attention network (RCAN) for image restoration. We demonstrate that UNet-RCAN achieves higher prediction accuracy compared to other state-of-the-art deep learning algorithms while maintaining the resolution of an output image compared to ground-truth data acquired with optical microscopes. We applied our method to three fluorescence imaging modalities. Firstly, we successfully demonstrate that UNet-RCAN can achieve up to two orders of magnitude acceleration in stimulated emission depletion (STED) imaging while maintaining super-resolution. This significant acceleration enables mitigation of photobleaching and photodamage by robust restoration of noisy 2D and 3D STED images from multiple targets as well as live-cell STED imaging of inner-mitochondrial dynamics with a ten-fold increase in the number of acquired frames compared to conventional STED microscopy. Secondly, we apply our approach in restoring high-resolution widefield deconvolution images of living cells with low light intensity and low photodamage. We show that the accuracy of deconvolution can significantly improve after image restoration with deep learning. Lastly, we show the application of UNet-RCAN in the resolution enhancement of single-shot volumetric imaging with a low numerical aperture objective lens.

Quantum dots (QDs), especially colloidal semiconductor QDs, possess properties including high quantum yields, narrow fluorescence spectra, broad absorption and excellent photostability, making them extremely powerful in bioimaging. In this thesis, we studied the fluorescence properties of QDs and attempted multiple ways to boost applications of QDs in bioimaging field. By time-correlated single photon counting (TCSPC) measurement, we quantitatively interpreted the fluorescence mechanism of colloidal semiconductor QDs. To enhance QD fluorescence, we used a porous alumina membrane as a photonic crystal structure to modulate QD fluorescence. We studied the acid dissociation of 3-mercaptopropionic acid (MPA) coated QDs mainly through electrophoretic mobility of 3-MPA coated CdSe QDs and successfully demonstrated the impact of pH change and Ca2+ ions. Blinking phenomena of both CdSe-CdS/ZnS core-shell QDs and 3C-SiC nanocrystals (NCs) were studied. A general model on blinking characteristics relates the on-state distribution to CdSe QD surface conditions. The energy relaxation pathway of fluorescence of 3C-SiC NCs was found independent of surface states. To examine QD effect on ciliated cells, we conducted a 70-day long experiment on the bioelectric and morphological response of human airway epithelial Calu-3 cells with periodic deposition of 3-MPA coated QDs and found the cytotoxicity of QDs was found very low. In a brief summary, our study of QD could benefit in bioimaging and biosensing. Especially, super-resolution fluorescent bioimaging, such as, stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM), may benefit from the modulation of the QD blinking in this study. And fluorescence lifetime imaging (FLIM) microscopy could take advantage of lifetime modulation based on our QD lifetime study. / <p>QC 20160905</p>

The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.

Exposure of hepatoma cell lines to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is serving as a model for a systems biological study concerning the response of cells to contaminant molecules. Several aspects of the cellular distribution of the aryl hydrocarbon receptor (AhR) and its ligand B[a]P have been addressed by different live cell imaging techniques: The intracellular distribution of the B[a]P/AhR complex is visualized by means of confocal laser scaning microscopy (cLSM) and the intracellular transport rates of the complex is investigated by fluorescence recovery after photobleaching (FRAP) technique. Furthermore, cLSM image stacks of living cells are generated for the modeling of three dimensional (3-D) cell geometries. In order to prevent photochemical damage of the living cells induced by UV excitation of B[a]P, visualization is done by B[a]P’s auto fluorescence using near infrared two-photon-excitation. Murine Hepatoma 1c1c7 cells are exposed to graded concentrations of B[a]P (50 nM to 20 μM) for different incubation time periods (15 minutes to 48 hours). The highest amounts of B[a]P were found in lipid droplets and lysosomes, where the B[a]P molecules are collected and form large aggregates. We were able to work with concentrations down to 50 nM corresponding to that used for genomic and proteomic investigations. Also, for the first time imaging of B[a]P metabolites inside lipid droplets is presented in this work. The data and the model developed in this study will provide new insights into the systematic regulation of the B[a]P, the AhR as well as the receptor-ligand-complex pathway and the study will also serve as a prototype for elucidating other stress response pathways in the future.

Traditional brain imaging modalities, for example, MRI, CT scan, X-ray, etc. can provide precise and high-resolution images of the brain for diagnosing lesions, tumors or clots inside the brain. However, these modalities require bulky and expensive test setups accessible only at specialized diagnostic centers, and hence may not be suitable or affordable to many patients. Furthermore, the inherent health risks limit the usability of these modalities for frequent monitoring. Microwave imaging is deemed a promising alternative due to its being cost-effective, portable, non-ionizing, non-intrusive. Therefore, this work aims to design an effective microwave head imaging system for the detection of blood clots inside the brain. Two miniaturized antipodal Vivaldi antenna designs are proposed which can provide wideband operation covering the low microwave frequency range (within 1 - 6 GHz) while having electrically small dimensions, directional radiation pattern with reasonable gain, and without requiring immersion in any matching/ coupling liquid. A head imaging system is presented which utilizes a quarter-head scanning approach, to reconstruct four images of the brain by scanning four quarters of the head, using the designed antipodal wideband Vivaldi antenna. A numerical brain model, with and without the presence of blood clot, is simulated using the proposed head-imaging system. At each quarter, the antenna is placed at nine different positions for scanning. The reflected signal at each position is processed and using confocal microwave imaging technique four images of the brain are reconstructed. A comparison is made among the four images in terms of their intensities, for the detection and approximate location of the blood clot inside the brain. The presence of higher intensity regions in any specific quarter of the head demonstrates the presence of a clot and its location and validates the feasibility of the proposed head imaging system using the low frequency wideband Vivaldi antenna.

The structural and functional development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential to understand in order to enable pharmaceutical testing, disease modeling, and ultimately therapeutic use. Recent developments in the field of bioengineering have led to improvements in the efficiency and efficacy of growth methods that allow hiPSC- CMs to be studied in greater detail. However, engineered cardiac tissue still has not achieved a level of maturation necessary for the majority of biomedical applications. Thus, new technologies and methods are necessary to realize the long-term benefits of engineered cardiac tissue. To better understand the development of the tissue, further characterization of the structure and function of these cardiomyocytes is required. In this work, we describe advances using a method not commonly applied to these materials, Small Angle X-ray Scattering (SAXS). SAXS was used to characterize the structural development of hiPSC- CMs on a 3D multicellular platform in their early stages of maturation. The myofilament lattice spacing was found to monotonically decrease as the tissue matured from its initial state post-seeding at a rate between 0.75 and 1 nm per day between days 3 and 10 of maturation. With 49 total samples across three different batches of tissue, the p value for correlation between the lattice plane spacing and maturation time was p<0.05, indicating a statistically significant correlation. In tests of the tissue response to fixation with varying doses of KCl relaxation buffer, results showed a general trend of decreased myofilament spacing with increasing KCl concentration. However, in the concentrations between 60mM and 120mM, a characteristic increase in spacing is observed. Beat force was also measured prior to measuring myofilament spacing and this resulted in a graphically suggestive correlation. However, ANOVA analysis results in a p value of 0.35 which is statistically insignificant. Finally, methods were tested to monitor the myofilament lattice spacing in contracting tissue and found no evidence of contraction-based changes in the myofilament lattice. / 2024-01-15T00:00:00Z

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(工学) / 甲第23915号 / 工博第5002号 / 新制||工||1781(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 大江 浩一, 教授 近藤 輝幸, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>Mechanical oscillating drills and saws are used in orthopaedic surgery to cut bone and develop screw-holes; however, their use causes friction resulting in significant thermal damage. Ultrashort pulsed lasers appear well-suited to replace traditional tools as they have the ability to efficiently remove bone tissue while causing only minimal collateral damage. Laser ablation also has the added advantages of: (i) no mechanical vibration; (ii) minimal invasiveness; and (iii) small focus spot size. In this thesis work, we experimentally investigated a few key aspects of ultrashort laser ablation of bone tissue.</p> <p>The ablation threshold of unaltered bone was measured using the <em>D</em>²technique and found to range from 1.66 J/cm²± 0.87 J/cm² to 2.37 J/cm²± 0.78 J/cm² depending on incident pulse number. The reduction in ablation threshold with pulse number was an indication of an incubation effect. Using a power law model, the incubation coefficient, ζ, was measured to be 0.89 ± 0.03.</p> <p>The effect of specific laser parameters and drilling protocols on ablation efficiency was also characterized. For ultrashort pulses (≤10 ps), the removal rate was found to be inversely related to the pulse duration; however, irradiation with 5-10 ps pulses were also shown to result in significant tissue removal. With a pulse repetition rate of 1 kHz, the removal rate was observed to be highest when ablating with 50-100 pulses per spot.</p> <p>Larger volumes (>1 mm³) of bone tissue were removed using laser scanning procedures. A series of scanned concentric circles produced a structure ~2.4 mm deep; however, ablated side-lobes were present at oblique angles to the incident beam. A two-layer structure subsequently produced no side-lobes. The ablative precision in trabecular bone was observed to be less than cortical bone. Using mimicked Nd:YAG laser parameters, cylindrical drilling produced craters significantly less deep than those achieved with a typical Ti:Sapphire configuration. The ability to drill large-scale holes using low average pulse energies and optimized scanning procedures will alleviate the stringent requirements for optical components in clinical practice.</p> / Master of Science (MSc)