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The everlasting battles between humans and bacteria entered a new stage with the discovery of antibiotics that saved millions of lives in the 20th century. However, the rise of antibiotic-resistant strains, often resistant to multiple classes of antibiotics, poses a renewed threat to human health. Compared to the quick and swift evolution of the resistant phenotypes, the development of new antibiotics has stagnated for decades, exacerbated by a limited understanding of the evolutionary mechanisms driving antibiotic resistance.Bacteria, thriving in diverse and harsh environments, have evolved myriad strategies to survive various stresses, including starvation, osmotic stress, heavy metals, antimicrobial agents, and so on. Understanding bacterial responses to these stresses is critical for delaying the emergence of antibiotic resistance mutations, reducing pathogenic bacterial tolerance to adverse environments, and optimizing industrial production processes.In this work, different tools were deployed to detect, visualize, quantify, and eventually predict bacterial behavior under different stresses. A widely used molecule resazurin was found to be an ideal candidate for a novel accurate, cheap, and highly effective bioassay that could monitor intracellular NADH level and bacterial stress response in real time. An algorithm depicting bacterial metabolite profile was developed to detect minor heavy metal contamination in the drinking water, harnessing bacteria as powerful indicators to pollution. Lastly, we adapted a novel model to study the persistent bacterial community known as biofilms. Biofilm attaches to biotic or abiotic surfaces, is resistant to adverse conditions, and causes great pain to our patients. We simulated the osmotic pressure perturbation in vivo to explain the mechanisms of cross-protection between different stresses and found inspiration to postulate new strategies to eliminate stubborn bacterial communities.

In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.

[Hypothesis] Response of lipid bilayers to external mechanical stimuli is an active area of research with implications for fundamental and synthetic cell biology. Developing novel tools for systematically imposing mechanical strains and non-invasively mapping out interfacial (membrane) stress distributions on lipid bilayers can accelerate research in this field., [Experiments] We report a miniature platform to manipulate model cell membranes in the form of droplet interface bilayers (DIBs), and non-invasively measure spatio-temporally resolved interfacial stresses using two photon fluorescence lifetime imaging of an interfacially active molecular flipper (Flipper-TR). We established the effectiveness of the developed framework by investigating interfacial stresses accompanying three key processes associated with DIBs: thin film drainage between lipid monolayer coated droplets, bilayer formation, and bilayer separation., [Findings] The measurements revealed fundamental aspects of DIBs including the existence of a radially decaying interfacial stress distribution post bilayer formation, and the simultaneous build up and decay of stress respectively at the bilayer corner and center during bilayer separation. Finally, utilizing interfacial rheology measurements and MD simulations, we also reveal that the tested molecular flipper is sensitive to membrane fluidity that changes with interfacial stress - expanding the scientific understanding of how molecular flippers sense stress.

Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments., Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood. We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood. Our findings revealed light-induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light-dependent electron transport. Younger stems exhibited higher light-induced O2 production and dark respiration rates than older ones. While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments.

Dental caries is the most widespread non-communicable disease affecting 2.5 billion people worldwide. Recently various optical spectroscopy methods have gained a lot of interest in dental research and caries detection due to their noninvasive and nonionizing nature. We demonstrate here, to the best of our knowledge, for the first time simultaneous Raman, fluorescence intensity and fluorescence lifetime imaging of carious human enamel with a single device based on a time-resolved single-photon avalanche diode (SPAD) sensor fabricated in 110-nm CMOS technology. The sensor has identical 256 spectral channels each of them containing an integrated tunable 7-bit time-to-digital converter (TDC) enabling time- and spectrally-resolved photon counting. We were able to show with the spectrometer built around the CMOS SPAD sensor and by utilizing a simple unsupervised machine learning algorithm (K-means) that a more comprehensive and reliable images of the enamel and caries-affected regions can be obtained by combining the information from the three different optical spectroscopy methods. We believe that the technology presented here could pave the way for novel multimodal optical devices not only in dental research and clinical dentistry but also in other biomedical applications in which molecular and chemical changes are studied.

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.

To meet the logistical needs of supply and demand for heme, organisms need to make it, move it to the right place, and degrade it when necessary. More specifically, the provision and distribution of heme must be coupled with the synthesis of heme proteins and meticulously regulated so that heme-tuned cellular pathways are only activated on demand. However, how cells handle heme to meet transient cellular requirements is yet to be clarified and represents a long-lasting question in bioinorganic chemistry. This study deploys a genetically encoded heme sensor (mAPXmEGFP) and Fluorescence Lifetime Imaging Microscopy to investigate the cellular mechanisms controlling heme bioavailability. The analysis is paired with the contextualisation of the data within the intricate network of regulation that oversees the levels of key proteins (i.e. ALAS1, HO-1, HO-2, and GAPDH) and their coordination with an intracellular heme buffering system that ensures heme homeostasis. Further, the implications and possible drawbacks deriving from the deployment of genetically encoded sensors in live cells is discussed and explored.

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. MOSAI

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report direct blind unmixing of lifetimes with subnanosecond resolution and subpixel spatial resolution using standard raster scan FLIM images. We demonstrate our method using a wide range of simulated and experimental data.

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. MOSAI

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report direct blind unmixing of lifetimes with subnanosecond resolution and subpixel spatial resolution using standard raster scan FLIM images. We demonstrate our method using a wide range of simulated and experimental data.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

The collagen VI-related disorders (COL6-RDs) are a group of severe congenital neuromuscular disorders with no available treatment. New insight into its molecular characterisation together with the advance in the microscopy field offers the opportunity of making early diagnosis a quotidian process. In this study, three state-of-the-art microscopy techniques such as light sheet fluorescence microscopy (LSFM), second-harmonic generation (SHG) microscopy and fluorescence lifetime imaging microscopy (FLIM) are used to study a mouse model of the disorder. With these techniques parameters that define the pathological phenotype such as the cross-sectional area (CSA) of the muscle fibres, the distance between the myosin bands within the sarcomeres, and the differential lifetimes of the muscles autofluorescence can be compared to the wild type.

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to temperature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid-ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different compositions in the 15–60 °C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520–550 nm and emission maxima at 580–640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospholipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 ± 0.010 ns), gel (1.16 ± 0.070 ns), and liquid-ordered (1.79 ± 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly in use in biophysical studies of membranes. According to temperature and composition, lipid bilayers can exist in liquid-crystalline (or liquid- disordered), liquid-ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different compositions in the 15-60ºC range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been used. Nile red shows excitation maxima at 520-550 nm and emission maxima at 580-640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquidordered > gel > rippled > liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospholipids. The probe is suitable for FLIM imaging studies of isolated membrane vesicles under the confocal microscope. Rotational relaxation times have been measured for Nile red in liquid-disordered (0.71 ns), gel (1.22 ns), and liquid-ordered (1.93 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to tem-perature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid -ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different composi-tions in the 15-60 degrees C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520-550 nm and emis-sion maxima at 580-640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospho-lipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 +/- 0.010 ns), gel (1.16 +/- 0.070 ns), and liquid-ordered (1.79 +/- 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.

In a recent work, we reported a methodology for imaging the different stages of amyloid aggregation in quantitative multiparametric dual-color fluorescence lifetime imaging (FLIM) and superresolution microscopy by using a pair of dyes capable of binding aggregates and undergoing subsequent intra-aggregate energy transfer (FRET) (RuizArias et al. Sensors Actuat. B, 2022, 350:130882). In this microarticle we present the optimization process for choosing the best pair of dyes through a screening of different naphthalimides and quinolimides and other known amyloid-binding dyes.

The presence of neuritic plaques and amyloid fibrils arising from the misfolding of certain proteins is the principal molecular indicator of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Methodologies for studying the early stages of amyloid aggregation are rapidly arising to provide a better understanding of the mechanism of fibrillization and cytotoxicity and to identify potential targets for diagnosis and therapy. The method presented here involves the simultaneous use of two different fluorophores, a quinolimide derivative and Nile Blue A. These are capable of interacting with and reporting on the formation of preamyloid aggregates and fibrils of apoferritin through fluorescence resonance energy transfer (FRET), which occurs between them, thus maximizing the contrast in detection and quantitative information of such amyloid species by using multidimensional fluorescence lifetime imaging microscopy (FLIM).

How could reproductive outcomes be improved? What could be the role of metabolomics in Assisted Reproduction? Increasing the effectiveness of procedures and treatments in reproductive medicine is one of the main keys to improvement. Currently, metabolomics is a hot research topic. Despite the different techniques with applications in daily clinical practice, the advances in AI and Systems Biology allow omics sciences to open new perspectives for the future. In this way, they could become a powerful complementary and non-invasive tool relevant to the study of male and female infertility, aimed at improving embryo selection and implantation, with the ultimate target of achieving a live and healthy newborn at home., ¿Cómo se podrían mejorar los resultados reproductivos? ¿Cuál podría ser el papel de la metabolómica en Reproducción Asistida? Aumentar la efectividad de los procedimientos y tratamientos en medicina reproductiva es una de las principales claves de mejora. Actualmente, la metabolómica es un tema de investigación candente. A pesar de las diferentes técnicas con aplicación en la práctica clínica diaria, los avances en Inteligencia Artificial (IA) y en Biología de sistemas permiten a las ciencias ómicas abrir nuevas perspectivas a futuro. De este modo, podrían convertirse en una poderosa herramienta complementaria y no invasiva relevante para el estudio de la infertilidad masculina y femenina, orientados hacia una mejora en la selección e implantación embrionaria, con el objetivo final de la consecución del recién nacido vivo y sano en casa.

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.

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.

The circular squeeze-film-bearing marvel shaped between upper strong impermeable circle and lower porous-unpleasant plate with the impacts of two roughness designs (radial and circumferential) on the porous surface utilizing FF lubricant. Here, the FF is constrained by diagonal radially factor magnetic field (VMF) as a result of acquiring preferred position of creating most extreme field at the required dynamic contact zone. Also, the VMF is considered in light of the fact that uniform magnetic field doesn't improve bearing exhibitions. The porous-unpleasant surface is considered in light of getting favorable position of self-greasing up property. Utilizing FF stream model by R. E. Rosensweig and roughness impact by Christensen's stochastic hypothesis adjusted Reynolds equation is determined, which is fathomed for load-conveying limit with respect to various shapes (exponential, secant, perfect representation of secant and parallel) of the upper circle. The outcomes are looked at among changed shapes and the effects of penetrability and roughness examples are considered. The motivation behind considering the present issue is lies in the perception that the investigations of the vast majority of various structures squeeze-films (circle plate, round and hollow plate, two- parallel plates, and so forth.) diminish to the present case or present case is the restricting instance of various structures squeeze-films.

Squeeze film geometry of truncated cone. Lower porous plate squeeze film orientation of various shapes (annular, round, elliptic, rectangular and cone) utilizing Morgan-Cameron guess. The impacts of the state of plate and porosity on the bearing execution are determined. The ferrofluid based squeeze film for round and conical orientation. The attractive field considered was the transverse way of the fluid flow. Here, they have considered Shliomis model to tackle the issue since it dealt with pivot of the fluid particles just as fluid. The subsequent overseeing conditions are nonlinear coupled conditions and is comprehended utilizing annoyance strategy regarding dimensionless Brownian unwinding time parameter. The impact of attractive fluid parameters on different bearing attributes is examined numerically. This article examined porous truncated cone squeeze film-bearing model thinking about the impacts of porosity, penetrability, squeeze speed and slanted variable attractive field. Impacts of two porousness models-globular circle and narrow gaps are additionally talked about. Articulations for pressure and burden conveying capacity are acquired. The outcomes for dimensionless burden conveying capacity are figured.

The circular squeeze-film-bearing marvel shaped between upper strong impermeable circle and lower porous-unpleasant plate with the impacts of two roughness designs (radial and circumferential) on the porous surface utilizing FF lubricant. Here, the FF is constrained by diagonal radially factor magnetic field (VMF) as a result of acquiring preferred position of creating most extreme field at the required dynamic contact zone. Also, the VMF is considered in light of the fact that uniform magnetic field doesn't improve bearing exhibitions. The porous-unpleasant surface is considered in light of getting favorable position of self-greasing up property. Utilizing FF stream model by R. E. Rosensweig and roughness impact by Christensen's stochastic hypothesis adjusted Reynolds equation is determined, which is fathomed for load-conveying limit with respect to various shapes (exponential, secant, perfect representation of secant and parallel) of the upper circle. The outcomes are looked at among changed shapes and the effects of penetrability and roughness examples are considered. The motivation behind considering the present issue is lies in the perception that the investigations of the vast majority of various structures squeeze-films (circle plate, round and hollow plate, two- parallel plates, and so forth.) diminish to the present case or present case is the restricting instance of various structures squeeze-films.

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.

On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ras nanocluster. However, it is still incompletely understood, in which physiological contexts nanoclustering is regulated and which constituents are parts of nanocluster. Nonetheless, disruption of nanoclustering faithfully diminishes Ras activity in cells, suggesting Ras nanocluster as potential drug targets.While there are several methods available to study Ras nanocluster , fluorescence or Förster resonance energy transfer (FRET ) between fluorescently labeled, nanoclustered Ras proteins is a relatively simple readout. FRET measurements using fluorescence lifetime imaging microscopy (FLIM ) have proven to be robust and sensitive to determine Ras nanoclustering changes. Loss of FRET that emerges due to nanoclustering reports on all processes upstream of Ras nanoclustering, i.e., also on proper trafficking or lipid modification of Ras. Here we report our standard FLIM-FRET protocol to measure nanoclustering-dependent FRET of Ras in mammalian cells. Importantly, nanoclustering-dependent FRET is one of the few methods that can detect differences between the Ras isoforms.

Naturally fluorescent polymeric molecules such as collagen, resilin, cutin, suberin, or lignin can serve as renewable sources of bioproducts. Theoretical physics predicts that the fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls that form woody biomass. Lignin is composed of different phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured the wood cell wall composition by classical analytical methods in these trees. Using statistical modeling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm (flowering) plants. In a first step toward tailoring lignin biosynthesis toward improvement of woody biomass feedstocks, we show how FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Naturally fluorescent polymeric molecules such as collagen, resilin, cutin, suberin, or lignin can serve as renewable sources of bioproducts. Theoretical physics predicts that the fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls that form woody biomass. Lignin is composed of different phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured the wood cell wall composition by classical analytical methods in these trees. Using statistical modeling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm (flowering) plants. In a first step toward tailoring lignin biosynthesis toward improvement of woody biomass feedstocks, we show how FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Naturally fluorescent polymeric molecules such as collagen, resilin, cutin, suberin, or lignin can serve as renewable sources of bioproducts. Theoretical physics predicts that the fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls that form woody biomass. Lignin is composed of different phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured the wood cell wall composition by classical analytical methods in these trees. Using statistical modeling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm (flowering) plants. In a first step toward tailoring lignin biosynthesis toward improvement of woody biomass feedstocks, we show how FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

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 circular squeeze-film-bearing marvel shaped between upper strong impermeable circle and lower porous-unpleasant plate with the impacts of two roughness designs (radial and circumferential) on the porous surface utilizing FF lubricant. Here, the FF is constrained by diagonal radially factor magnetic field (VMF) as a result of acquiring preferred position of creating most extreme field at the required dynamic contact zone. Also, the VMF is considered in light of the fact that uniform magnetic field doesn't improve bearing exhibitions. The porous-unpleasant surface is considered in light of getting favorable position of self-greasing up property. Utilizing FF stream model by R. E. Rosensweig and roughness impact by Christensen's stochastic hypothesis adjusted Reynolds equation is determined, which is fathomed for load-conveying limit with respect to various shapes (exponential, secant, perfect representation of secant and parallel) of the upper circle. The outcomes are looked at among changed shapes and the effects of penetrability and roughness examples are considered. The motivation behind considering the present issue is lies in the perception that the investigations of the vast majority of various structures squeeze-films (circle plate, round and hollow plate, two- parallel plates, and so forth.) diminish to the present case or present case is the restricting instance of various structures squeeze-films.

Squeeze film geometry of truncated cone. Lower porous plate squeeze film orientation of various shapes (annular, round, elliptic, rectangular and cone) utilizing Morgan-Cameron guess. The impacts of the state of plate and porosity on the bearing execution are determined. The ferrofluid based squeeze film for round and conical orientation. The attractive field considered was the transverse way of the fluid flow. Here, they have considered Shliomis model to tackle the issue since it dealt with pivot of the fluid particles just as fluid. The subsequent overseeing conditions are nonlinear coupled conditions and is comprehended utilizing annoyance strategy regarding dimensionless Brownian unwinding time parameter. The impact of attractive fluid parameters on different bearing attributes is examined numerically. This article examined porous truncated cone squeeze film-bearing model thinking about the impacts of porosity, penetrability, squeeze speed and slanted variable attractive field. Impacts of two porousness models-globular circle and narrow gaps are additionally talked about. Articulations for pressure and burden conveying capacity are acquired. The outcomes for dimensionless burden conveying capacity are figured.

On the plasma membrane, Ras is organized into laterally segregated proteo-lipid complexes called nanoclusters. The extent of Ras nanoclustering correlates with its signaling output, positioning nanocluster as dynamic signaling gain modulators. Recent evidence suggests that stacked dimers of Ras and Raf are elemental units at least of one type of Ras nanocluster. However, it is still incompletely understood, in which physiological contexts nanoclustering is regulated and which constituents are parts of nanocluster. Nonetheless, disruption of nanoclustering faithfully diminishes Ras activity in cells, suggesting Ras nanocluster as potential drug targets.While there are several methods available to study Ras nanocluster , fluorescence or Förster resonance energy transfer (FRET ) between fluorescently labeled, nanoclustered Ras proteins is a relatively simple readout. FRET measurements using fluorescence lifetime imaging microscopy (FLIM ) have proven to be robust and sensitive to determine Ras nanoclustering changes. Loss of FRET that emerges due to nanoclustering reports on all processes upstream of Ras nanoclustering, i.e., also on proper trafficking or lipid modification of Ras. Here we report our standard FLIM-FRET protocol to measure nanoclustering-dependent FRET of Ras in mammalian cells. Importantly, nanoclustering-dependent FRET is one of the few methods that can detect differences between the Ras isoforms.

Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.

We present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale.

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach.

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular lev-el. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron or even na-noscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. Wear debris chemical and biological impact on the surrounding periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis was suc-cessfully characterized by applying a combination of photon, electron and ion beam mi-cro-spectroscopy techniques, that includes hybrid optical fluorescence and reflectance mi-cro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris was found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosomes. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at the molecular and subcellular level contributes to a better understanding of the mac-roscopic inflammatory processes observed on the tissue level. The established label-free correla-tive microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.

Naturally fluorescent polymeric molecules such as collagen, resilin, cutin, suberin, or lignin can serve as renewable sources of bioproducts. Theoretical physics predicts that the fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls that form woody biomass. Lignin is composed of different phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured the wood cell wall composition by classical analytical methods in these trees. Using statistical modeling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm (flowering) plants. In a first step toward tailoring lignin biosynthesis toward improvement of woody biomass feedstocks, we show how FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Naturally fluorescent polymeric molecules such as collagen, resilin, cutin, suberin, or lignin can serve as renewable sources of bioproducts. Theoretical physics predicts that the fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls that form woody biomass. Lignin is composed of different phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured the wood cell wall composition by classical analytical methods in these trees. Using statistical modeling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm (flowering) plants. In a first step toward tailoring lignin biosynthesis toward improvement of woody biomass feedstocks, we show how FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised hierarchical-lineage model of homeostasis.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a fu

Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised "hierarchical-lineage" model of homeostasis.

Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast. Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm). Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expec

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a fu

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.