京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18587号 / 工博第3948号 / 新制||工||1607(附属図書館) / 31487 / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 北條 正樹, 教授 北村 隆行, 教授 琵琶 志朗 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Confocal microscopy demonstrated its value in the diagnosis of Acanthamoeba and fungal keratitis which considered sight-threatening corneal diseases. However, it can be difficult to find and train confocal microscopy graders to accurately detect Acanthamoeba cysts and fungal filaments in the images. Use of an automated system could overcome this problem and help to start the correct treatment more quickly. Also, response to treatment can be difficult to assess in infectious keratitis using clinical examination alone, but there is evidence that the morphology of filaments and cysts may change over time with the use of correct treatment. An automated system to analyse confocal microscopy images for such changes would also assist clinicians in determining whether the ulcer is improving, or whether a change of treatment is needed. This research proposes a fully automated novel system with GUI to detect cysts and hyphae (filaments) and measure useful quantitative parameters for them through many stages; Image enhancement, image segmentation, quantitative analysis for detected cysts and hyphae, and registration and tracking of ordered sequence of images. The performance of the proposed segmentation procedure is evaluated by comparing between the manual and the automated traced images of the dataset that was provided by the Manchester Royal Eye Hospital. The positive predictive values rate of cysts for Acanthamoeba images was 76%. For detected hyphae in Fusarium images, many standard measurements were computed. The accuracy of their values was quantified by calculating the percent error rate for each measurement and which ranged from 23% to 49%.

Yes / Background and Objective Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. Methods First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). Results The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland–Altman plot shows that 95% of the data are between the 2SD agreement lines. Conclusions We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image.

Yes / Endothelial dysfunction and damage underlie cerebrovascular disease and ischemic stroke. We undertook corneal confocal microscopy (CCM) to quantify corneal endothelial cell and nerve morphology in 146 patients with an acute ischemic stroke and 18 age-matched healthy control participants. Corneal endothelial cell density was lower (P<0.001) and endothelial cell area (P<0.001) and perimeter (P<0.001) were higher, whilst corneal nerve fbre density (P<0.001), corneal nerve branch density (P<0.001) and corneal nerve fbre length (P=0.001) were lower in patients with acute ischemic stroke compared to controls. Corneal endothelial cell density, cell area and cell perimeter correlated with corneal nerve fber density (P=0.033, P=0.014, P=0.011) and length (P=0.017, P=0.013, P=0.008), respectively. Multiple linear regression analysis showed a signifcant independent association between corneal endothelial cell density, area and perimeter with acute ischemic stroke and triglycerides. CCM is a rapid non-invasive ophthalmic imaging technique, which could be used to identify patients at risk of acute ischemic stroke. / Qatar National Research Fund Grant BMRP20038654

Fluorescence microscopy has played a pivotal role in the realm of biological and biomedical research, allowing researchers to delve into the intricacies of living organisms at the cellular and molecular levels. By using fluorescent probes, one can visualize specific molecules and structures within cells, fundamentally transforming our comprehension of biology and medicine. However, fluorescence microscopy faces its own set of challenges, namely, photobleaching and photodamage. Photobleaching involves the irreversible loss of fluorescence signal during imaging, while photodamage results in harmful effects on cells. Both severely limit fluorescence signal and observation time. Although remedies exist to mitigate these problems, most of them rely on chemical approaches. In this dissertation, to address these issues, I investigated two optical approaches that exploit control of light either in space or time. Firstly, I developed multiline scanning confocal microscopy (mLS) with a digital micro-mirror device. This method provides programmable patterns of the illumination beam as well as the detection slit. Through experimental results and optical simulations, I assessed the depth discrimination of mLS under different optical parameters and compared it with a multipoint system such as spinning disk confocal microscopy (SDCM). Surprisingly, under the same illumination duty cycle, I found that mLS offers better optical sectioning than SDCM. Importantly, the parallelized line illumination showed a much lower photobleaching rate compared to single-line scanning microscopy, while their optical sectioning capabilities remained similar. I applied this technique to visualize heterogeneous mouse epiblast stem cells, a challenging task in imaging. Secondly, I delved into low photobleaching rate two-photon microscopy (2PM). 2PM inherently provides excellent optical sectioning due to its nonlinear effects, making it suitable for high-resolution imaging within biological tissues. However, the high peak power of ultrafast pulses has always been associated with severe photobleaching, posing a longstanding challenge. I found that controlling the repetition rates of ultrafast lasers is a potential strategy to enhance photostability. Specifically, I used repetition rates lower or higher than 80 MHz in 2PM and conducted systematic experiments to investigate how optical parameters such as wavelengths, excitation powers, and pulse schemes can influence the photobleaching kinetics of fluorescent proteins and organic fluorophores. This thesis embarks on a journey to explore innovative strategies and methodologies aimed at reducing photobleaching while maintaining high-quality imaging in the realm of fluorescence microscopy.

The normal aging process in humans is characterized by a number of hallmark changes including decreased white matter volume in the brain and accompanying cognitive decline. This is in contrast to neurodegenerative aging processes which involve acute pathology which results in neuronal cell death. Studying non-degenerative normal aging in humans can be difficult because of the high prevalence of neurodegenerative diseases in the population and other potentially confounding effects. Rhesus monkeys are an excellent model organism for the study of normal aging, as their aging process has been demonstrated to involve diminished white matter volume, but they do not suffer from neurodegenerative diseases such as Alzheimer's. In this study we seek to quantify levels of myelin degradation using confocal microscopy in regions of interest where it has been previously demonstrated that loss of white matter integrity results in lower levels of cognitive function across different treatment groups including aging monkeys, calorie restricted monkeys, and controls for calorie restricted monkeys. These areas include prefrontal white matter which is vital to executive function, the hippocampus which is integral to memory consolidation and the learning process, and finally the anterior, middle, and posterior cingulum bundle. The cingulum bundle contains a diverse variety of projections between cortical and subcortical regions, including but not limited to projections to and from the cingulate cortex which has been demonstrated to be vital for emotional processing, the limbic system, and a wide spectrum of other functions. We aim to quantify white matter degradation in these regions by using immunofluorescent tagging for healthy myelin basic protein (MBP) and degraded myelin basic protein (dMBP) and by measuring the colocalization between the two. For prefrontal white matter and hippocampus, we did not find significant differences in myelin degradation across treatment groups. In the cingulum bundle, however, we did find a significant effect of treatment on overall myelin damage throughout the bundle, and in particular we determined that there was a significant difference in colocalization in the anterior cingulum bundle between aging monkeys and control calorie restricted monkeys. Analysis of behavioral testing data yielded surprising results as we were unable to find a strong correlation between our measure for myelin degradation, and level of cognitive impairment. Our results indicate that there are likely differences in regional vulnerability to age related myelin damage across different white matter regions of the brain, however we would like to expand on this study to gain a more accurate understanding of how loss of white matter volume is distributed through the brain and the impact that has on cognitive outcomes.

The purpose of this study was to use retrograde tracing techniques to examine hepatic neuroanatomy in the rat model, with special emphasis upon the identification of previously undiscovered intrahepatic parasympathetic ganglia. Retrograde analysis was performed using Fluoro-Gold (FG) tracer injections of both male and female Sprague-Dawley rats. To accurately examine the neural connectivity of both the vasculature and the parenchyma, the FG-labelled livers were divided into two groups. In the first, vessel trees were extracted via dissection and whole-mounted for bright field and confocal visualization. Left lateral lobes taken from the male and female liver that constituted the second group were sectioned, and slices from various layers of tissue were fixed to slides and visualized. The results indicated the presence of several large, fluorescent structures bearing a strong resemblance to parasympathetic ganglia. However, the images were not detailed enough to properly differentiate true ganglia from similar paraganglia. Regardless, the importance of this experiment lies in its attempt to revisit an understudied field in neuroscience, and the findings of this study could potentially provide a starting point for further inquiry.

Optical microscopy is the oldest form of microscopy that has been visually aiding scientific research. In our research, I have reported here two such optical microscopy techniques for two different projects. In the first project, we re-developed an instrumentation of a cost-effective, high-performing, single-molecular localization super-resolution microscopy setup that breaks the diffraction limitation barrier. Then we use a stochastic image capturing technique to capture the best precision image positions of gold nanoparticles. In our second project, we apply confocal microscopy technique to image DNA molecular nanoscale structural alterations of chromatin in cell nuclei of gut tissues caused by total body irradiation (TBI). We then quantify these alterations using a light localization technique called inverse participation ratio (IPR) using the confocal micrographs of the sample. Our results show radiation causes reduction and saturation of DNA spatial mass density fluctuations that were observed for different durations of post-irradiation.

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