Developing Photoacoustic Tomography Devices for Translational Medicine and Basic Science Research

Photoacoustic (PA) tomography (PAT) provides volumetric images of biological tissue with scalable spatial resolutions and imaging depths, while preserving the same imaging contrast—optical absorption. Taking the advantage of its 100% sensitivity to optical absorption, PAT has been widely applied in structural, functional, and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. Intuitively, hemoglobin has been the most commonly studied biomolecule in PAT due to its strong absorption in the visible wavelength regime. One of the main focuses of this dissertation is to investigate an underexplored wavelength regime—ultraviolet (UV), which allows us to image cell nuclei without labels and generate histology-like images naturally from unprocessed biological tissue. These preparation-free and easy-to-interpret characteristics open up new possibilities for PAT to become readily applicable to other important biomedical problems (e.g., surgical margin analysis, Chapter 2) or basic science studies (e.g., whole-organ imaging, Chapter 3). For instance, we developed and optimized a PA microscopy system with UV laser illumination (UV-PAM) to achieve fast, label-free, multilayered, and histology-like imaging of human breast cancer in Chapter 2. These imaging abilities are essential to intraoperative surgical margin analysis, which enables promptly directed re-excision and reduces the number of repeat surgeries. We have incorporated the Grüneisen relaxation (GR) effect with UV-PAM to improve the performance of our UV-PAM system (e.g., the axial resolution), thus providing more accurate three-dimensional (3D) information (Chapter 4). The nonlinear PA signals caused by the GR effect enable optical sectioning capability, revealing important 3D cell nuclear distributions and internal structures for cancer diagnosis. In the final focus of this dissertation, we have implemented a low-cost PA computed tomography (PACT) system with a single xenon flash lamp as the illumination source (Chapter 5). Lasers have been commonly used as illumination light sources in PACT. However, lasers are usually expensive and bulky, limiting their applicability in many clinical usages. Therefore, the use of a single xenon flash lamp as an alternative light source was explored. We found that PACT images acquired with flash lamp illumination were comparable to those acquired with laser illumination. This low-cost and portable PACT system opens up new potentials, such as low-cost skin melanoma imaging in undeveloped countries

Quantitative Image Processing for Three-Dimensional Episcopic Images of Biological Structures: Current State and Future Directions

Episcopic imaging using techniques such as High Resolution Episcopic Microscopy (HREM) and its variants, allows biological samples to be visualized in three dimensions over a large field of view. Quantitative analysis of episcopic image data is undertaken using a range of methods. In this systematic review, we look at trends in quantitative analysis of episcopic images and discuss avenues for further research. Papers published between 2011 and 2022 were analyzed for details about quantitative analysis approaches, methods of image annotation and choice of image processing software. It is shown that quantitative processing is becoming more common in episcopic microscopy and that manual annotation is the predominant method of image analysis. Our meta-analysis highlights where tools and methods require further development in this field, and we discuss what this means for the future of quantitative episcopic imaging, as well as how annotation and quantification may be automated and standardized across the field

Platform for High-Throughput Testing of the Effect of Soluble Compounds on 3D Cell Cultures

In vitro 3D culture could provide an important model of tissues in vivo, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique (“cells-in-gels-in-paper”, CiGiP) that permits rapid assembly of arrays of 3D cell cultures and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual “spots” (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes. A custom-made 96-well holder isolated the cell-containing zones from each other. Each well contained media to which a different compound could be added. After culture and disassembly of the holder, peeling the layers apart “sectioned” the individual 3D cultures into 200-μm-thick sections which were easy to analyze using 2D imaging (e.g., with a commercial gel scanner). This 96-well holder brings new utilities to high-throughput, cell-based screening, by combining the simplicity of CiGiP with the convenience of a microtiter plate. This work demonstrated the potential of this type of assays by examining the cytotoxic effects of phenylarsine oxide (PAO) and cyclophosphamide (CPA) on human breast cancer cells positioned at different separations from culture media in 3D cultures.Chemistry and Chemical Biolog

Using strain field mining to reveal the spatial distributions of tensile, fatigue, and fracture damage accumulation in paper

The most common nonwoven fiber composite material, paper, has a porous, heterogeneous fiber network structure and complicated mechanical properties. The mechanical properties of commercial, machine made papers are orthotropic and are sensitive to loading rate, moisture content, and temperature. Thus, defining the constitutive relationship of paper has remained as a challenge due to the stochastic nature of the structure and countless variables that affect the mechanics of paper. Moreover, the technology to non-destructively characterize the three-dimensional network topography at the fiber length scale is not readily available. This presents a critical barrier to establishing the structure-property relationships of paper. Here, I approached the problem with a fundamentally different strategy and used the structure of the strain fields as a proxy for the network topography. The strain fields of paper from tensile, fatigue, tearing experiments revealed new information about each damage mechanisms. During the tensile deformation, the interplay between the axial and the transverse motions in the fiber network resulted in specimen-orientation-dependent (MD and CD) parameters such as Poisson's ratio, hot spot length scales, and the degree of nonaffinity, D. These metrics were direct manifestation of the anisotropic fiber network in paper. Next, I used strain field mining to track the fatigue crack lengths and quantified crack growth rates during cyclic and constant loading conditions. The fracture profiles and the crack growth rates revealed that there was a unique fatigue damage mechanism in paper which induced the fiber fracture rather than the fiber pull-out. Moreover, I found that the pre-applied creep damage in paper can significantly reduce the fatigue crack growth rate and extend paper's high cycle fatigue life. Lastly, from the strain fields of tearing specimens, I was able to characterize paper's crack tip process zone and the zone of active plasticity (ZAP) whose shape depended on the orientation of the fiber network. Although paper has a completely different structure and failure mechanism from metals, I found that tearing of paper also followed a steady-state process, which was previously observed in thin sheet aluminum foils.Ph.D

Occurrence and fate of micro- and nanoplastic in the terrestrial environment

The worldwide production of plastic has grown exponentially since the 1950’s and revolutionized our daily life. Simultaneously, plastic pollution in the environment has become a global issue and micro- (MP) and nanoplastics (NP) have now been detected even in the most remote ecosystems. There is currently a data gap due to a lack of analytical methods on the occurrence and characterisation of two highly relevant categories of plastic in the soil environment: tire wear particles (TWP), which concentration in the environment is expected to be high and carry toxic additives, and NP, which toxicity has been demonstrated on soil organisms and is characterized by its ability to cross cell membranes. The effects of micro- and nanoplastic (MNP) on their surrounding environment are determined by their size, morphology, surface characteristics and chemical composition, which can be affected by soil residence time. As the soil is often considered as sink for MNP, it is crucial to investigate and understand the different weathering factors which might affect the MNP properties. To address these knowledge gaps, three main objectives were identified in the scope of this study: develop an extraction and single particle identification method for the quantification and characterisation of (i) TWP and (ii) NP in soil samples and (iii) characterise the physico-chemical properties at the surface of plastic debris occurring in the soil environment, as well as assess the effect of soil and UV weathering as single ageing factors. In order to realise the first objective, a method of extraction and identification of TWP in soil samples based on their black colour was developed using optical microscopy. Cryo-grinded TWP down to a size of 35 μm could be detected with a >85% but the tests conducted with environmental TWP showed that the density used in this study was not efficient to separate the whole range of TWP occurring in different densities. Yet, TWP concentration in highway adjacent soil samples ranged between 8084 ±1059 and 2562 ± 1160 TWP kg-1 dry soil and showed similar trends and magnitude order than previously reported concentrations. Thus, the developed protocol was estimated sufficiently accurate for TWP monitoring in soil samples. Regarding the second objective, an extraction and identification method for NP in soil samples was developed using X-ray spectro-microscopy (STX-NEXAFS). The results demonstrated the suitability of the technique for the imaging and chemical characterisation of individual NP with a minimum dimension of ≈100 nm and its application to the analysis of pure NP and for NP present in environmental and food matrices. However, it was not possible to obtain quantitative data on the NP present in the samples, as the method was too time consuming to allow the measurement of a high number of particles. For the last objective, STXM-NEXAFS was applied to the characterisation of the surface alterations of natural-soil weathered, soil-incubated and UV exposed polymers. A surface alteration on a depth varying between 150 and 1000 nm on could be observed and the analysis of the replicate’s measurement acquired on the same plastic debris highlighted the heterogeneity of the processes affecting polymers surface. The comparison of UV weathered and natural-soil weathered samples showed that the two treatments led to different surface alterations and the absence of surface alteration after one-year soil incubation indicated slow aging of polymers in this medium. Moreover, the very first step of surface fragmentation was observed on a PS fragment, providing an insight on the factors and processes leading to the release of MP and NP in soils. Overall, the present research contributed significantly to the development of innovative methods to characterise MNP in the soil environment. The results obtained helped to provide ground information on the characteristic of environmental MP and NP, which is of high importance to design ecotoxicological test using environmentally relevant material as well as validate predictive models to better understand the potential risk that MP and NP represent for the ecosystems

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery

Flexible CO₂ sensor architecture with selective nitrogen functionalities by one-step laser-induced conversion of versatile organic ink

Nitrogen-doped carbons (NC) are a class of sustainable materials for selective CO2 adsorption. We introduce a versatile concept to fabricate flexible NC-based sensor architectures for room-temperature sensing of CO2 in a one-step laser conversion of primary coatings cast from abundant precursors. By the unidirectional energy impact in conjunction with depth-dependent attenuation of the laser beam, a layered sensor heterostructure with porous transducer and active sensor layer is formed. Comprehensive microscopic and spectroscopic cross-sectional analyses confirm the preservation of a high content of imidazolic nitrogen in the sensor. The performance was optimized in terms of material morphology, chemical composition, and surface chemistry to achieve a linear relative resistive response of up to ∆R/R0 = -14.3% (10% of CO2). Thermodynamic analysis yields ΔadsH values of -35.6 kJ·mol-1 and 34.1 kJ·mol-1 for H2O and CO2, respectively. The sensor is operable even in humid environments (e.g., ∆R/R0,RH=80% = 0.53%) and shows good performance upon strong mechanical deformation

Probing the Unseen Depths of the Hepatic Microarchitecture via Multimodal Microscopy

Multimodal microscopy combines the advantages and strengths of different imaging modalities in order to holistically characterise the organisation of biological organisms and their comprising constituents under healthy and diseased conditions, down to the spatial resolution required to understand the morphology and function of such structures. Given the profound advantages conferred by such an approach, this work broadly aimed to develop and exploit various multimodal and multi-dimensional imaging modalities in a complimentary, combined and/or correlative manner – namely, three-dimensional scanning electron microscopy, transmission electron tomography, bright-field light microscopy, confocal laser scanning microscopy and X-ray micro-computed tomography – in order to characterise and collect new information on the normal and pathological microarchitecture of rodent and human liver tissue in 3-D under various experimental conditions. The data reported in this work includes a comparative analysis of a variety of sample preparation protocols applied to rat liver tissue to determine the suitability of such protocols for the application of serial block-face scanning electron microscopy (SBF-SEM). Next, 3-D modelling and morphometric analysis (utilising the premier SBF-SEM protocol) was performed in order to visualise and quantify key features of the hepatic microarchitecture. We further outline a large-volume correlative light and electron microscopy approach utilising selective molecular probes for confocal laser scanning microscopy (actin, lipids and nuclei), combined with the 3-D ultrastructure of the same structures of interest, as revealed by SBF-SEM (Chapter 2). Development of a straightforward combinatorial sample preparation approach, followed by a swift multimodal imaging approach – combining X-ray micro-computed tomography, bright-field light microscopy and serial section scanning electron microscopy – facilitated the cross correlation of structure-function information on the same sample across diverse length scales (Chapter 3). Next, we outline a novel “silver filler pre-embedding approach” in order to reduce artefactual charging, minimise dataset acquisition time and improve resolution and contrast in rat liver tissue prepared for SBF-SEM (Chapter 4). Next, we employ a complementary imaging approach involving serial section scanning electron microscopy and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in human patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation (Chapter 5). Finally, the significance of the results obtained, and major scientific advances reported in this work are discussed in-depth against the relevant literature. This is proceeded by the future outlooks and research that remains to be done, followed by the main conclusions of this Ph.D thesis (Chapter 6). In summary, our findings firmly establish the immense importance and value of contemporary multimodal microscopy modalities in modern life science research, for holistically revealing cellular structures along the vast length scales amongst which they exist, under healthy and clinically relevant pathological conditions