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Pancreatic adenocarcinoma (PDAC) is an aggressive disease that is typically diagnosed at a late stage. PDAC development occurs over approximately 20 years and the initiating event is often an activating mutation in key cell signalling hub KRAS. Despite extensive research into PDAC and KRAS, the impact of both genetic and non-genetic variability during the earliest steps of carcinogenesis are scantly described. We hypothesise that both forms of heterogeneity might alter the efficiency of PDAC initiation. On the one hand, oncogenic KRAS alleles might rewire cellular signalling and metabolic networks causing phenotypes and adaptation in an allelespecific manner. On the other hand, cell-to-cell variability within mutant clones might permit a population of cells to escape tumour suppressive mechanisms such as senescence and adapt to oncogenic signalling, therefore supporting early cancer development. We have combined cell genetics, single-cell biochemistry and a multiomics approach to investigate both genetic and non-genetic heterogeneity during initiation of PDAC. We generated an isogenic panel of cell lines harbouring the three most common mutations of KRAS in PDAC (G12D, G12R, G12V). This system consisted of inducible expression of KRAS mutants in immortalized but non-transformed pancreatic cells to mimic PDAC initiation. In Chapter 2, we explore the short-term cellular response to KRAS induction and adaptation of cells to oncogenic KRAS. Using western blotting, metabolomics and RNAseq we show how specific KRAS mutant alleles influence the cell signalling, nucleotide metabolism and cell morphology. We show that the G12D and G12V mutants, frequently observed in KRAS-driven cancers, differ significantly from the PDAC-specific G12R mutant. The mutants display allele-specific growth factor signalling, purine metabolism rewiring and an epithelial to mesenchymal transition. In Chapter 3, we looked in greater temporal resolution and at a single-cell level at the immediate signalling and metabolic response to KRAS mutations, focusing mainly on G12D. We used a FRET sensor reporting mitochondrial ATP levels in live cells to study G12D cells at multiple time points. We found an early drop in mitochondrial ATP which was only identified when considering intracellular heterogeneity. This drop was followed by a peak in mitochondrial ATP at a later time point, caused by a combination of purine synthesis and mitochondrial ATP production. We also observed an increase in activated and total levels of mitochondrial dynamics protein DRP1 and the presence of an anti-oxidant signature. The involvement of DRP1 suggests an increase in mitochondrial homeostasis early during adaptation to oncogenic KRAS. Using techniques such as metabolomics, phosphoflow and scRNAseq we were able to further dissect this adaptation process. We also found heterogeneity in cells exploring broader levels of phosphorylated ERK upon G12D induction, which further varied over the studied time course. The recorded variability of pERK levels supports the hypothesis of an oncogenic signalling sweetspot. Taken together, this study sheds light on the process of adaptation to oncogenic KRAS. Adaptation to mutant KRAS occurs over multiple stages even within this phase of early adaptation. These stages include variation in pERK signalling, nucleotide metabolism and likely mitochondrial homeostasis. Our results suggest that both genetic and non-genetic heterogeneity play important roles in these processes and thus, the earliest events of PDAC initiation. Understanding signalling and metabolic rewiring in a KRAS mutant specific manner may help us untangle allele-specific dependencies that could inform patient stratification and disease management. Furthermore, identifying processes by which normal cells can adapt to oncogenic KRAS at a single-cell level reveals the necessary steps for disease initiation, and may provide options for preventative intervention.

For those suffering from cancer nanoparticle-enhanced radiotherapy has shown great potential as a means to improve patient outcome. In order to maximise the benefit obtained from such treatment a full understanding of the uptake dynamics of nanoparticles is required. As such the aim of this body of work is to conceptualise and experimentally demonstrate novel approaches to probe the uptake of gold nanoparticles (AuNPs) in cancer cells. In the first study a technique is developed to probe the temporal dynamics of AuNP uptake by performing multiphoton fluorescence-lifetime imaging microscopy (MP-FLIM) on live cells. Using this technique and subsequent analysis methods multiple datasets were obtained showing the association of AuNPs with the cell membrane in real-time. This study then goes on to provide a means to quantify this behaviour. The second study details the development of a novel X-ray fluorescence microscopy technique to investigate the uptake of AuNPs spatially on a sub-cellular level. Particularly, an emphasis is placed on providing a means to unambiguously determine if nuclear uptake of AuNPs has taken place. Using this technique this study reports the first unambiguous evidence of individually resolved AuNPs within an intact cell nucleus.

Understanding and identifying critical protein-protein interactions is just one of the key outcomes in biological research. It can help to confirm key cellular interactions, which in some fields, such as cancer research, can result in a greater understanding of disease pathogenesis, elucidate mechanisms of therapeutic resistance and aid in the development of new specific targets, leading to new methods of prevention and treatment. Time-correlated single photon counting fluorescence lifetime imaging microscopy is just one of the tools used to carry out this line of research. Here we demonstrate a direct interaction between two proteins involved in gene regulation and expression; p21 and FMN2. Furthermore, we also show the capability of this system to measure chromatin compaction in three dimensions. However, fluorescence lifetime imaging has some drawbacks, acquisition times on such a system can range from the tens of seconds to minutes, which is often too long to comprehensively measure many biological events. But microscopy is always developing, aided by new techniques and, perhaps even more so, new technological developments. This thesis also demonstrates two new methods of light sheet microscopy, that use both new equipment made available because of technological developments to allow time resolved imaging and traditional microscopic aspects to form a light sheet system based on polarisation. It outlines the design and how to build these systems and presents their function to show their great promise. Both techniques presented in this thesis utilise aspects of light not conventionally used in light sheet microscopy. Further development of these systems and application of emerging technologies will yield a system capable of outperforming current light sheet fluorescence microscopy-based fluorescence lifetime imaging techniques. The implementation of polarisation control into such a system would enable three-dimensional anisotropy based SPIM-FLIM measurements, an indispensable tool in researching molecular orientation and mobility at a macroscopic level in developing organisms.

Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.

FLIM is branch of microscopy mainly used in biology which is quickly improving thanks to a rapid enhancement of instrumentation and techniques enabled by new sensors. In FLIM, the most precise method of measuring fluorescent decays is called TCSPC. High voltage PMT detection devices together with costly and bulky optical setups which scan the sample are usually required in TCSPC instrumentation. SPADs have enabled a big improvement in TCSPC measurement setup, providing a CMOS compatible device which can be designed in wide arrays format. However, sensors providing in-pixel TCSPC do not scale in size and in large array like the time-gated SPAD pixel sensors do. Time-gated pixels offer a less precise lifetime estimation, discarding any photon information outside a given time window, but this loss in photon-efficiency is offset by gains in pixel size. This work is aimed at the development of a wide field TCSPC sensor with a pixel size and fill factor able to reduce the cost of such devices and to obtain a high resolution time-resolved fluorescence image in the shortest time possible. The study focuses on SPAD and pixel design required to maximise the fill factor in sub 10 μm pixel pitch. Multiple pixel designs are proposed in order to reduce pixel area and so enable affordable wide array TCSPC systems. The first proposed pixel performs the CMM lifetime estimation in order to reduce the frame rate needed to stream the data out of the SPAD array. This pixel is designed in a 10 μm pitch and attains with the most aggressive design a fill factor of 10:17 %. A second design proposes an analogue TCSPC which consists in a S/H TAC circuitry. This simpler pixel can achieve a higher fill factor of 19:63% as well as smaller pitch of 8 μm thanks to the adoption of SPAD n-well and electronics area sharing. This last design is implemented in a 320 x 256 SPAD array in which is included part of a novel ADC aimed at reduction of the processing time required to build a TCSPC histogram. A more conventional analogue readout is used to evaluate the pixel performance as well as a more fine TCSPC histogram. The device was used to measure the fluorescence lifetime of green micro-spheres while the 2b flash ADC is used to demonstrate rapid resolution and separation of two different fluorescence decays.

Many of the wastes associated with the nuclear fuel cycle are toxic to the biosphere; advancing the use of high resolution spectroscopy applied to these materials will provide the chemical speciation of the interaction between nuclear waste and geological material, improving confidence in a permanent disposal method and informing clean-up operations. Luminescence spectroscopy of uranyl(VI) is a well-established technique for the molecular speciation of uranium-mineral interactions. This work explores the use of both micro- and macroscopic luminescence spectroscopy to expose uranyl(VI) speciative heterogeneity in a range of minerals which have been exposed to uranyl(VI) salt solutions. A comprehensive review of the available literature on the interaction of uranyl(VI) with a range of geological media is assessed and compared. The review finds considerable ambiguity in the speciation of uranyl(VI) at the mineral water interface. A database reporting the multi parametric luminescence properties of uranyl(VI) with silica gel, quartz, bayerite, boehmite, muscovite, kaolinite and montmorillonite (SWy-2 and STx-1b) is presented and discussed. Although some of the results are consistent with previously reported values, many newly identified species are reported and their identification speculated. Parallel factor analysis is used to deconvolute the excitationemission matrix of uranyl(VI) sorbed to silica gel between pH 3 and pH 10. The results are used to identify the spectroscopic properties of complexes >(SiO)2UO2 and >(SiO)2UO2OH and thus new complexation coefficients (log(K)) for their formation with the silica gel surface are determined, log(K1) = 9.22 ± 0.02 and log(K2) = 3.45 ± 0.01, respectively. The investigation also provides insight into the fundamental properties of uranyl(VI) excitation pathways, which are not yet fully understood. Confocal microscopy and phosphorescent lifetime image mapping (PLIM) is used to expose the sub-micron heterogeneity of uranyl(VI) sorption complexation across mineral surfaces of silica gel, bayerite and montmorillonite (STx-1b). The results suggest that changes in the uranyl(VI) lifetime can be used to observe and understand submicron changes in uranyl(VI) complexation at hitherto unknown temporal resolution.

The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.

The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.

I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.

The transfer of mitochondria, whether through natural or artificial pathways, has been experimentally validated across various cell lines, demonstrating potential benefits for host cells experiencing mitochondrial dysfunction. However, conflicting reports suggest that our understanding of the outcomes of mitochondria transfer in host cells remains incomplete. This dissertation aims to delve into the concept of mitochondria transfer and examine its effects on metabolism, viability, and oxidative stress in breast cancer host cells. The novelty of this dissertation lies in the utilization of fluorescence lifetime imaging to analyze the metabolic profiles of individual mitochondria. Through the combination of fluorescence labeling and NADH lifetime imaging, we have discovered that newly formed mitochondria in breast cancer host cells exhibit an increase in the bound fraction of NADH, indicating a reliance on Oxidative Phosphorylation (OXPHOS). While the enhancement of OXPHOS leads to heightened respiration and proliferation, the accumulation of reactive oxygen species (ROS) levels surpasses the tolerance threshold of breast cancer cells towards oxidative stress, resulting in elevated drug sensitivity when exposed to anticancer drugs. The significance of this dissertation resides in its multifaceted approach, shedding light on the impact of mitochondria transfer and establishing new methodologies for future research endeavors.

Multiplexed mRNA and protein profiling in the spatial context provides important new information enabling basic research and clinical applications. Unfortunately, most existing spatial transcriptomics and proteomics methods are limited due to either low multiplexing or assay complexity. Here, we introduce a new spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based target decoding. This technology is the first application combining the biophotonic techniques, Spectral and Fluorescence Lifetime Imaging and Microscopy (FLIM), to the field of spatial transcriptomics and proteomics. By integrating the time dimension with conventional spectrum-based measurements, MOSAICA enables direct and highly multiplexed in situ spatial biomarker profiling in a single round of staining and imaging while providing error correction removal of background autofluorescence. We demonstrated MOSAICA’s capabilities in cell culture and Formalin-Fixed Paraffin-Embedded (FFPE) tissues while obtaining a strong correlation with sequencing data (Pearson’s r = 0.96). We then demonstrate simultaneous co-detection of protein and mRNA in colorectal cancer cells. To answer biological questions with a simple 3-4 plex immunofluorescence panel, we developed a low-cost Tissue Imager for under $9,000 and achieved a performance on par with commercial fluorescence microscopes that cost ~20x more. Additionally, another tool we developed to study the tissue microenvironment was cell-based mechanosensors to quantitively and dynamically assess the tissue mechanics. We have already used MOSAICA to study colon cancer heterogeneity, profile neurological mRNA panels in brain tissue, and profile immuno-oncology panels for skin tissue. MOSAICA represents a simple, versatile, and scalable tool for targeted spatial transcriptomics and proteomics analysis that can find broad utility in constructing human cell atlases, elucidating biological and disease processes in the spatial context, and serving as companion diagnostics for stratified patient care.

Profiling multiomic biomarkers in bulk and in situ provides critical information which enables basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims to develop two core technologies that address 1) bulk profiling issues with sensitivity and low throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection (IC3D). This integrated approach combines microfluidic droplet partitioning technology, fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic biomarkers from minimally processed blood samples. To address the second issue, this work introduces a new spatial multi-omics technology termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and time-resolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and spectral components simultaneously within the same pixel or image of a sample to enable highly increased multiplexing capabilities with standard optical systems. Overall, these two technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive, and/or multiplexed protein/transcriptomic analysis.

Metastasis remains the leading cause of cancer mortality but its mitochondrial dysregulation - from its morphology and motility to its metabolic influence - in modulating cancer’s progression is poorly understood. This dissertation describes the development and use of complex microphysiological culture systems to place cancer in its proper environmental context for mitochondrial phenotypic discoveries. FLIM of NADH is used to probe the metabolic differences between invasive and non- or less-invasive cancer cells and elucidate potential invasion-specific therapeutic targets. This dissertation also introduces Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer finds that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, Mitometer shows that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is then applied to investigate cellular contractility in the influence of mitochondrial phenotype. The goal of this dissertation as a whole is to accentuate the importance of creating new assays and techniques with the impact of context and translatability in mind.

The binding of antibody and antigen constitutes the basis of immunoassays. In this thesis, we aim to explore this binding through three perspectives: a) improving the kinetics of the reaction by increasing mass transport, b) developing a novel method to study the kinetics of binding, and c) using immunoassays for quantifying surface antigen on tumors to uncover tumor heterogeneity. Nowadays, immunoassays are mostly performed on surfaces, which facilitates washing steps and exchanging reagents. A key limitation of such assays is the formation of a layer depleted of analyte, which reduces the reaction rate and thus decreases the overall sensitivity of the test. In the first part of this thesis, we explore the most common current strategies to reduce the depletion layer, i.e., the use of shakers. We show that while shakers are easy to use, they often offer unpredictable results. Such results can be greatly improved through the use of microfluidics, which offers the possibility to control mass transport through convective flows, improving signal while reducing total assay time. Nevertheless, microfluidics has not been adapted to the most common substrates used for immunoassays, microtiter plates. Thus, we propose a new microfluidic concept for controlling the mass transport in microtiter plate wells, which through the use of different kinetic zones allows the exploration of samples with high dynamic ranges in a single test. We then develop a method to evaluate the kinetics of antibody binding using fluorescence lifetime imaging microscopy. Such a method allows a characterization of kinetic constants by comparing the fraction of bound to unbound antibodies on a variety of substrates. We demonstrate its use in breast cancer derived cell blocks and in cancer tissues. Finally, we explore the heterogeneity in cancer, i.e., the different phenotypical profiles that cells in cancer show. Studying tumor heterogeneity is critical to understand cancer evolution in a patient and thus be able to perform accurate patient stratification. We develop a method easily implementable in pathology labs that uses the kinetics of binding of antibodies through immunohistochemical stains to quantify the presence of antigens on different regions of a sample. By performing this localized quantification, we can also explore the heterogeneity present on the tissue. We further investigate heterogeneity using a workflow involving the local tissue lysis and antibody microarray analysis. This method shows a higher capacity of multiplexing, which allowed us to investigate the relative variations of 13 proteins and their inter and intra-tumoral heterogeneity, highlighting the presence of multiple phenotypical variants in a single patient. Thus, in this thesis, we show several use scenarios of antibody-antigen binding kinetics, which highlight the potential of this simple binding to provide us with powerful techniques.

Choroidal melanocytes are the melanin-producing cells in the vascular uveal tract of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment and display cellular functions, and extracellular interactions that are biologically distinct from skin melanocytes. Melanocytes (and melanins) are important in normal human eye physiology, with roles including photoprotection, regulation of oxidative damage and potentially immune regulation. Our current understanding of the regional distribution, spatial melanin composition and melanosome distribution in choroidal melanocytes (and melanocytic lesions) is primarily informed by immunolabelled eye tissues, or by melanin quantification following tissue-destructive techniques. To address these knowledge gaps, the thesis initially quantified the regional distribution of human choroidal melanocytes (HCMs) in histology eye sections. The distribution of HCMs was statistically preserved in central (sub-foveal and near the optic disc), sub-perifoveal and far peripheral (near the ora serrata) choroidal regions. The quest for understanding eye melanocytes continued next with the visualisation of pigmented HCMs using specific melanocyte-related target proteins, fluorescent immunohistochemistry (IHC) and melanin bleaching in post-mortem tissue. A combined fluorescent-IHC and 5% H2O2 melanin bleaching protocol provided a balance between fluorescence signal detection from target proteins of dark pigmented HCMs and tissue integrity including HCM cytomorphology. However, melanin bleaching did incur loss of the fluorescence emission spectral and lifetime signatures of the cytoplasmic melanins. As such, melanins (eumelanin and pheomelanin) and melanosomes in label-free cultured and whole tissue HCMs were ultimately profiled and spatially visualised using non-invasive 2-photon microscopy (2PM) fluorescence spectral and lifetime acquisition and phasor plot segmentation. This approach successfully and consistently confirmed intracellular melanins as endogenous fluorophores, and as quantifiable and reliable biomarkers for variably pigmented HCMs. As a proof-of-concept, the 2PM FLIM acquisition and phasor plot analysis methods were used to define the melanin profiles in a small series of human choroidal melanocytic lesions.

Alcohol consumption has been recognized as a risk factor for breast cancer. It is positively correlated with the progression of breast cancer. Ethanol is studied to shift the metabolism of breast cancer cells. According to the Warburg Effect, cancer cells predominantly produce their energy through a high rate of glycolysis. I hypothesized that with acute ethanol exposure the breast cancer cells will shift their metabolism from oxidative phosphorylation to glycolysis. The metabolic shift of cancer cells changes the ratio of free and protein bound NADH, which is an essential coenzyme in cellular metabolic pathway. As such, investigating the fluorescent lifetime of free and protein bound NADH of breast cancer cells through Fluorescence Lifetime Imaging Microscopy (FLIM), I report the preliminary results on the metabolic shifts of two breast cancer cell lines: MDA-MB-231 and MCF-7 due to acute alcohol exposure.

Collagens and extra cellular matrix (ECM) proteins play important roles in the structural composition of tissues, and their modifications are implicated in diseases including cancer metastasis, tissue fibrosis, diabetic ulcers. Current tissue collagen analyses are based on destructive histological methods, whereas the ECM proteins’ intrinsic autofluorescences can provide a label-free detection and imaging to differentiate tissue compositions, modifications, and disease states. Fluorescence lifetime spectroscopy has been demonstrated as a key technique to distinguish ECM proteins in tissue. However, the large, expensive, and often operationally challenging instrumentation required to measure fluorescent lifetimes from tissues prevents the technique from gaining traction in biomedical applications.To this end, this thesis represents three major efforts to address this roadblock in tissue lifetime instrumentation by 1) demonstrate a facile fluorescence lifetime technique via frequency domain analysis, 2) realizing a hand-held multispectral lifetime probe for point detection on the skin, 3) realizing a low-cost, low complexity LED based imaging system to enable FLIM (fluorescence lifetime imaging microscopy) for histology without daunting instrumentation.In the first effort, the frequency domain (FD) fluorescence lifetime technique was demonstrated for the differentiation of collagens I/III and elastin, three major biophotonic components in wound tissues that undergo change during healing. In the second effort, a compact multispectral xsystem demonstrated the FD lifetime’s utility as a point-of-care device, and added additional wavelength-specific lifetime spectroscopy to further improve differentiation. The third effort expands theFD technique to an imaging modality, by leveraging LED scanning optomechanics to create histological images of tissues without labeling. Additionally, the scanning of a diabetic ulcer showed evidence that the technique can provide quantitative information on disease modification of the tissue that current immunohistochemical methods are ill-equipped to provide. By providing a quantitative, more nuance model of tissue biophotonics while using low-cost, low complexity FD instrumentation, the works in this thesis hopes to increase accessibility and popularity of label-free ECM lifetime spectroscopy for a number of biomedical and clinical applications.

Reduced nicotinamide adenine dinucleotide (NADH) is a major cofactor for a large number of biological enzymes that are essential in a myriad of metabolic pathways such as glycolytic and oxidative phosphorylation pathways. In addition, NADH is intrinsically fluorescent and therefore has the potential of serving as a biomarker to monitor mitochondrial dysfunctions associated with aging, cancer, and apoptosis. In this thesis, we investigate how macromolecular crowding may affect the biochemical reaction kinetics of NADH interaction with lactate dehydrogenase (LDH) as a model system in biomimetic crowding (e.g., Ficoll-enriched buffer at 0 ̶ 400 g/L). Using noninvasive, quantitative two-photon fluorescence lifetime and associated anisotropy, we exploit the sensitivities of NADH fluorescence lifetime and rotational diffusion to protein binding. To differentiate between viscosity and crowding effects on the reaction kinetics, we also conducted complementary measurements in glycerol-enriched buffer. Additionally, we are investigating the sensitivity of cellular NADH interaction with dehydrogenases to metabolic manipulations. Our quantitative and non-invasive methodology complements the traditional biochemical and thermodynamics techniques without the destruction of live cells. Intracellular NADH also exists as a mixture of free and enzyme-bound populations at dynamic equilibrium throughout living cells, which can be imaged using fluorescence lifetime imaging for both quantitative and noninvasive assessment of cellular metabolism. 2P-fluorescence lifetime imaging microscopy (FLIM) and 2P- fluorescence anisotropy of intrinsic NADH was measured in cultured mouse embryonic cells under both resting conditions and metabolic-manipulation.

Oxidative stress underlies the mechanisms of many diseases such as Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, diabetes, Multiple Sclerosis, bipolar disorder, and chronic fatigue syndrome. Oxidative stress is caused by excessive reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals, which are byproducts of oxygen metabolism in aerobic organisms. Environmental factors such as exposure to ultraviolet light, chemicals ingested by the diet, ionizing radiation, and cigarette smoke can also lead to production of ROS and therefore oxidative stress. As a result, there is a growing need for quantitative and noninvasive methodologies to probe oxidative stress at the single-cell level and ultimately in vivo. In this thesis, we examined the potential of natural coenzyme nicotinamide adenine dinucleotide (NADH) as a natural biomarker for oxidative stress in C3H 10T1/2 living cells in culture. NADH (fluorescent) is a coenzyme that is essential for energy metabolism via oxidative phosphorylation pathway in the inner membrane of mitochondria, which is also a major source for intrinsic ROS species generated through the electron transport chain. Our experimental multiparametric approach combined cell culture with fluorescence microscopy (confocal and two-photon) and spectroscopy (fluorescence lifetime imaging and time-resolved anisotropy) methods. Cultured cells were treated with hydrogen peroxide and rotenone in order to trigger oxidative stress. As a point of reference, conventional oxidative stress assays such as MitoSOX Red, JC-1, and H2DCFDA were used to optimize the chemical dosage and incubation time needed for observable oxidative stress. Our results help in the collective effort to establish cellular NADH autofluorescence as a natural biomarker for cellular metabolic health and oxidative stress.