New implementations of phase-contrast imaging

Phase-contrast imaging is a method of imaging widely used in biomedical research and applications. It is a label-free method that exploits intrinsic differences in the refractive index of different tissues to differentiate between biological structures under analysis. The basic principle of phase-contrast imaging has inspired a lot of implementations that are suited for different applications. This thesis explores multiple novel implementations of phase-contrast imaging in the following order. 1, We combined scanning Oblique Back-illumination Microscope (sOBM) and confocal microscope to produce phase and fluorescence contrast images in an endomicroscopy configuration. This dual-modality design provides co-registered, complementary labeled and unlabeled contrast of the sample. We further miniaturized the probe by dispensing the two optical fibers in our old design. And we presented proof of principle demonstrations with ex-vivo mouse colon tissue. 2, Then we explored sOBM-based phase and amplitude contrast imaging under different wavelengths. Hyperspectral imaging is achieved by multiplexing a wide-range supercontinuum laser with a Michaelson interferometer (similar to Fourier transform spectroscopy). It features simultaneous acquisition of hyperspectral phase and amplitude images with arbitrarily thick scattering biological samples. Proof-of-principle demonstrations are presented with chorioallantoic membrane of a chick embryo, illustrating the possibility of high-resolution hemodynamics imaging in thick tissue. 3, We focused on increasing the throughput of flow cytometry with principle of phase-contrast imaging and compressive sensing. By utilizing the linearity of scattered patterns under partially coherent illumination, our cytometer can detect multiple objects in the same field of view. By utilizing an optimized matched filter on pupil plane, it also provides increased information capacity of each measurement without sacrificing speed. We demonstrated a throughput of over 10,000 particles/s with accuracy over 91% in our results. 4, A fourth part, which describes the principle and preliminary results of a computational fluorescence endomicroscope is also included. It uses a numerical method to achieve sectioning effect and renders a pseudo-3D image stack with a single shot. The results are compared with true-3D image stack acquired with a confocal microscope

Harnessing label-free Raman spectroscopy for metastatic cancer diagnosis and biologic development

Optical spectroscopy is unique amongst experimental techniques in that it can be performed in near-physiological conditions, achieve high molecular specificity, and explore dynamics on timescales ranging from nanoseconds to days. In particular, Raman spectroscopy has emerged in the last two decades as a uniquely versatile method to investigate the structures and properties of molecules in diverse environments through interpreting vibrational transitions. In this thesis, we present four interconnected biomedical and biopharmaceutical applications of Raman spectroscopy that exploit its exquisite molecular specificity, non-perturbative nature, and near real-time measurement capability. In the first presented study, we harness spontaneous Raman spectroscopy in conjunction with multivariate analysis to rapidly and quantitatively determine antibody-drug conjugate aggregation with the goal of eventual application as an in-line tool for monitoring protein particle formation. By exploring subtle, but consistent, differences in spectral vibrational modes of various monoclonal antibodies (mAb) aggregations, a support vector machine-based regression model is developed which is able to accurately predict a wide range of protein aggregation. In addition, the investigation of these spectral vibrational modes also offers new insights into mAb product-specific aggregation mechanisms. Second, leveraging surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR), we present a design of plasmonic nanostructures based on rationally structured metal-dielectric combinations, which we call composite scattering probes (CSP). Specifically, we design CSP configurations that have several prominent resonance peaks enabling higher tunability and sensitivity for self-referenced multiplexed analyte sensing. The CSP prototypes were used to demonstrate differentiation of subtle changes in refractive index (as low as 0.001) as well as acquire complementary untargeted plasmon-enhanced Raman measurements from the biospecimen’s compositional contributors. In the third study, we demonstrate that Raman spectroscopy offers vital biomolecular information for early diagnosis and precise localization of breast cancer-colonized bone alterations. We show that as early as two weeks after intracardiac injections of breast cancer cells in mouse models, Raman measurements in femur and spine uncover consistent changes in both bone matrix and mineral composition. This research effort opens the door for improved understanding of breast metastatic tumor-related bone remodeling and establishing a non-invasive tool for detection of early metastasis and prediction of fracture risk. In parallel with this effort, we also seek to identify the differences between organ-specific isogenic metastatic breast cancer cells. By interpreting the informative spectral bands, we are able to unambiguously identify these isogenic cell lines as unique biological entities. Our spectroscopic study and corresponding metabolic research indicate that tissue-specific adaptations generate biomolecular alterations on cancer cells

Early brain activity : Translations between bedside and laboratory

Neural activity is both a driver of brain development and a readout of developmental processes. Changes in neuronal activity are therefore both the cause and consequence of neurodevelopmental compromises. Here, we review the assessment of neuronal activities in both preclinical models and clinical situations. We focus on issues that require urgent translational research, the challenges and bottlenecks preventing translation of biomedical research into new clinical diagnostics or treatments, and possibilities to overcome these barriers. The key questions are (i) what can be measured in clinical settings versus animal experiments, (ii) how do measurements relate to particular stages of development, and (iii) how can we balance practical and ethical realities with methodological compromises in measurements and treatments.Peer reviewe

Advanced methods in reproductive medicine: Application of optical nanoscopy, artificial intelligence-assisted quantitative phase microscopy and mitochondrial DNA copy numbers to assess human sperm cells

Declined fertility rate and population is a matter of serious concern, especially in the developed nations. Assisted Reproductive Technologies (ART), including in vitro fertilization (IVF), have provided great hope for infertility treatment and maintaining population growth and social structure. With the help of ART, more than 8 million babies have already been born so far. Despite the worldwide expansion of ART, there is a number of open questions on the IVF success rates. Male factors for infertility contribute equally as female factors, however, male infertility is primarily focused on the “semen quality”. Therefore, the search of new semen parameters for male fertility evaluation and the exploration of the optimal method of sperm selection in IVF have been included among the top 10 research priorities for male infertility and medically assisted reproduction. The development of imaging systems coupled with image processing by Artificial Intelligence (AI) could be the revolutionary step for semen quality analysis and sperm cell selection in IVF procedures. For this work, we applied optical nanoscopy technology for the analysis of human spermatozoa, i.e., label-based Structured Illumination Microscopy (SIM) and non-invasive Quantitative Phase Microscopy (QPM). The SIM results demonstrated a prominent contrast and resolution enhancement for subcellular structures of living sperm cells, especially for mitochondria-containing midpiece, where features around 100 nm length-scale were resolved. Further, non-labeled QPM combined with machine learning technique revealed the association between gradual progressive motility loss and the morphology changes of the sperm head after external exposure to various concentrations of hydrogen peroxide. Moreover, to recognize healthy and stress-affected sperm cells, we applied Deep Neural Networks (DNNs) to QPM images achieving an accuracy of 85.6% on a dataset of 10,163 interferometric images of sperm cells. Additionally, we summarized the evidence from published literature regarding the association between mitochondrial DNA copy numbers (mtDNAcn) and semen quality. To conclude, we set up the high-resolution imaging of living human sperm cells with a remarkable level of subcellular structural details provided by SIM. Next, the morphological changes of sperm heads resulting from peroxidation have been revealed by QPM, which may not be explored by microscopy currently used in IVF settings. Besides, the implementation of DNNs for QPM image processing appears to be a promising tool in the automated classification and selection of sperm cells during IVF procedures. Moreover, the results of our meta-analysis showed an association of mtDNAcn in human sperm cells and semen quality, which seems to be a relevant sperm parameter for routine clinical practice in male fertility assessment

An ex vivo system to study cellular dynamics underlying mouse peri-implantation development

マウスの着床期の胚発生を三次元で再現することに成功. 京都大学プレスリリース. 2022-02-09.Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures the study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to E6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of the cases, but its compatibility with light-sheet microscopy also enables the study of cellular dynamics through automatic cell segmentation. We find that, upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis, and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D ex vivo system thus offers unprecedented access to peri-implantation development for in toto monitoring, measurement, and spatiotemporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues

Developing novel fluorescent probe for peroxynitrite: implication for understanding the roles of peroxynitrite and drug discovery in cerebral ischemia reperfusion injury

Session 7 - Oral PresentationsSTUDY GOAL: Peroxynitrite (ONOO‐) is a cytotoxic factor. As its short lifetime, ONOO‐ is hard to be detected in biological systems. This study aims to develop novel probe for detecting ONOO‐ and understand the roles of ONOO‐ in ischemic brains and drug discovery ABSTRACT: MitoPN‐1 was found to be a ONOO‐ specific probe with no toxicity. With MitoPN‐1, we studied the roles of ONOO‐ in hypoxic neuronal cells in vitro and MCAO …postprin

Seeing the Big Picture: System Architecture Trends in Endoscopy and LED-Based hyperspectral Subsystem Intergration

Early-stage colorectal lesions remain difficult to detect. Early development of neoplasia tends to be small (less than 10 mm) and flat and difficult to distinguish from surrounding mucosa. Additionally, optical diagnosis of neoplasia as benign or malignant is problematic. Low rates of detection of these lesions allow for continued growth in the colorectum and increased risk of cancer formation. Therefore, it is crucial to detect neoplasia and other non-neoplastic lesions to determine risk and guide future treatment. Technology for detection needs to enhance contrast of subtle tissue differences in the colorectum and track multiple biomarkers simultaneously. This work implements one such technology with the potential to achieve the desired multi-contrast outcome for endoscopic screenings: hyperspectral imaging. Traditional endoscopic imaging uses a white light source and a RGB detector to visualize the colorectum using reflected light. Hyperspectral imaging (HSI) acquires an image over a range of individual wavelength bands to create an image hypercube with a wavelength dimension much deeper and more sensitive than that of an RGB image. A hypercube can consist of reflectance or fluorescence (or both) spectra depending on the filtering optics involved. Prior studies using HSI in endoscopy have normally involved ex vivo tissues or xiv optics that created a trade-off between spatial resolution, spectral discrimination and temporal sampling. This dissertation describes the systems design of an alternative HSI endoscopic imaging technology that can provide high spatial resolution, high spectral distinction and video-rate acquisition in vivo. The hyperspectral endoscopic system consists of a novel spectral illumination source for image acquisition dependent on the fluorescence excitation (instead of emission). Therefore, this work represents a novel contribution to the field of endoscopy in combining excitation-scanning hyperspectral imaging and endoscopy. This dissertation describes: 1) systems architecture of the endoscopic system in review of previous iterations and theoretical next-generation options, 2) feasibility testing of a LED-based hyperspectral endoscope system and 3) another LED-based spectral illuminator on a microscope platform to test multi-spectral contrast imaging. The results of the architecture point towards an endoscopic system with more complex imaging and increased computational capabilities. The hyperspectral endoscope platform proved feasibility of a LED-based spectral light source with a multi-furcated solid light guide. Another LED-based design was tested successfully on a microscope platform with a dual mirror array similar to telescope designs. Both feasibility tests emphasized optimization of coupling optics and combining multiple diffuse light sources to a common output. These results should lead to enhanced imagery for endoscopic tissue discrimination and future optical diagnosis for routine colonoscopy