Дослідження впливу пігментних параметрів на спектральні характеристики природних водних середовищ для задач екологічного контролю

Досліджено вплив співвідношень між основними пігментами, а саме хлорофілом a, хлорофілом b та каротиноїдами на спектральні характеристики водних середовищ з частинками фітопланктону. Розраховано спектральні характеристики показника розсіювання, фактору анізотропії, ефективних показників розсіювання та ослаблення, частки світла, розсіяного в передню півсферу, ймовірності виживання фотону, показника послаблення в малокутовому наближенні, коефіцієнту направленого пропускання та коефіцієнту дифузного відбиття для одного шару водного середовища з заданими структурними та пігментними параметрами фітопланктону, а також спектральну характеристику загального коефіцієнту дифузного відбиття на поверхні водного середовища.Исследовано влияние соотношений между основными пигментами, а именно хлорофиллом a, хлорофиллом b и каротиноидами на спектральные характеристики водных сред с частицами фитопланктона. Рассчитаны спектральные характеристики показателя рассеяния, фактора аниз отропии, эффективных показателей рассеяния и ослабления, части светового излучения рассеянного в переднюю полусферу, вероятности выживания фотона, показателя ослабления в малоугловом приближении, коэффициента направленного пропускания и коэффициента диффузного отражения для одного слоя водной среды с заданными структурными и пигментными параметрами фитопланктона, а также спектральную характеристику общего коэффициента диффузного отражения на поверхности водной среды.The influence of the relationships between the main pigments, namely chlorophyll a, chlorophyll b and carotenoids on the spectral characteristics of aqueous media with phytoplankton particles, was studied. The spectral characteristics of the scattering index, the anisotropy factor, theeffective scattering and attenuation parameters, the part of the light scattered into the forward hemisphere, the probability of photon survival, the attenuation index in the small-angle approximation, the transmission coefficient and the diffuse reflection coefficient for one layer of an aqueous medium with specified structural and pigment parameters of phytoplankton, as well as spectral characteristics of the total coefficient of diffuse reflection on the surface one medium

Tissue clearing

Tissue clearing of gross anatomical samples was first described more than a century ago and has only recently found widespread use in the field of microscopy. This renaissance has been driven by the application of modern knowledge of optical physics and chemical engineering to the development of robust and reproducible clearing techniques, the arrival of new microscopes that can image large samples at cellular resolution and computing infrastructure able to store and analyse large volumes of data. Many biological relationships between structure and function require investigation in three dimensions, and tissue clearing therefore has the potential to enable broad discoveries in the biological sciences. Unfortunately, the current literature is complex and could confuse researchers looking to begin a clearing project. The goal of this Primer is to outline a modular approach to tissue clearing that allows a novice researcher to develop a customized clearing pipeline tailored to their tissue of interest. Furthermore, the Primer outlines the required imaging and computational infrastructure needed to perform tissue clearing at scale, gives an overview of current applications, discusses limitations and provides an outlook on future advances in the field

Non-Invasive Photoacoustic Imaging of In Vivo Mice with Erythrocyte Derived Optical Nanoparticles to Detect CAD/MI.

Coronary artery disease (CAD) causes mortality and morbidity worldwide. We used near-infrared erythrocyte-derived transducers (NETs), a contrast agent, in combination with a photoacoustic imaging system to identify the locations of atherosclerotic lesions and occlusion due to myocardial-infarction (MI). NETs (≈90 nm diameter) were fabricated from hemoglobin-depleted mice erythrocyte-ghosts and doped with Indocyanine Green (ICG). Ten weeks old male C57BL/6 mice (n = 9) underwent left anterior descending (LAD) coronary artery ligation to mimic vulnerable atherosclerotic plaques and their rupture leading to MI. 150 µL of NETs (20 µM ICG,) was IV injected via tail vein 1-hour prior to photoacoustic (PA) and fluorescence in vivo imaging by exciting NETs at 800 nm and 650 nm, respectively. These results were verified with histochemical analysis. We observed ≈256-fold higher PA signal from the accumulated NETs in the coronary artery above the ligation. Fluorescence signals were detected in LAD coronary, thymus, and liver. Similar signals were observed when the chest was cut open. Atherosclerotic lesions exhibited inflammatory cells. Liver demonstrated normal portal tract, with no parenchymal necrosis, inflammation, fibrosis, or other pathologic changes, suggesting biocompatibility of NETs. Non-invasively detecting atherosclerotic plaques and stenosis using NETs may lay a groundwork for future clinical detection and improving CAD risk assessment

2D and 3D high-speed multispectral optical imaging systems for in-vivo biomedical research

Functional optical imaging encompasses the use of optical imaging techniques to study living biological systems in their native environments. Optical imaging techniques are well-suited for functional imaging because they are minimally-invasive, use non ionizing radiation, and derive contrast from a wide range of biological molecules. Modern transgenic labeling techniques, active and inactive exogenous agents, and intrinsic sources of contrast provide specific and dynamic markers of in-vivo processes at subcellular resolution. A central challenge in building functional optical imaging systems is to acquire data at high enough spatial and temporal resolutions to be able to resolve the in-vivo process(es) under study. This challenge is particularly highlighted within neuroscience where considerable effort in the field has focused on studying the structural and functional relationships within complete neurovascular units in the living brain. Many existing functional optical techniques are limited in meeting this challenge by their imaging geometries, light source(s), and/or hardware implementations. In this thesis we describe the design, construction, and application of novel 2D and 3D optical imaging systems to address this central challenge with a specific focus on functional neuroimaging applications. The 2D system is an ultra-fast, multispectral, wide-field imaging system capable of imaging 7.5 times faster than existing technologies. Its camera-first design allows for the fastest possible image acquisition rates because it is not limited by synchronization challenges that have hindered previous multispectral systems. We present the development of this system from a bench top instrument to a portable, low-cost, modular, open source, laptop based instrument. The constructed systems can acquire multispectral images at >75 frames per second with image resolutions up to 512 x 512 pixels. This increased speed means that spectral analysis more accurately reflects the instantaneous state of tissues and allows for significantly improved tracking of moving objects. We describe 3 quantitative applications of these systems to in-vivo research and clinical studies of cortical imaging and calcium signaling in stem cells. The design and source code of the portable system was released to the greater scientific community to help make high-speed, multispectral imaging more accessible to a larger number of dynamic imaging applications, and to foster further development of the software package. The second system we developed is an entirely new, high-speed, 3D fluorescence microscopy platform called Laser-Scanning Intersecting Plane Tomography (L-SIPT). L-SIPT uses a novel combination of light-sheet illumination and off-axis detection to provide en-face 3D imaging of samples. L-SIPT allows samples to move freely in their native environments, enabling a range of experiments not possible with previous 3D optical imaging techniques. The constructed system is capable of acquiring 3D images at rates >20 volumes per second (VPS) with volume resolutions of 1400 x 50 x 150 pixels, over a 200 fold increase over conventional laser scanning microscopes. Spatial resolution is set by choice of telescope design. We developed custom opto-mechanical components, computer raytracing models to guide system design and to characterize the technique's fundamental resolution limits, and phantoms and biological samples to refine the system's performance capabilities. We describe initial applications development of the system to image freely moving, transgenic Drosophila Melanogaster larvae, 3D calcium signaling and hemodynamics in transgenic and exogenously labeled rodent cortex in-vivo, and 3D calcium signaling in acute transgenic rodent cortical brain slices in-vitro

Towards multimodal nonlinear microscopy in clinics

Multimodal nonlinear microscopy combining two photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) represents a promising and powerful tool for biomedical diagnostics. The method enables label-free visualization of morphology and chemical composition of complex tissues as well as disease related changes and is as such as detailed as staining histologic methods. In this work a compact microscope utilizing novel fiber laser sources and a new approach for data analysis based on colocalization have been developed and tested for detecting various disease patterns, e.g., atherosclerosis and brain tumors.Mit Hilfe der nichtlinearen Multikontrast-Mikroskopie basierend auf den Prozessen Zweiphotonenfluoreszenz (TPEF), Frequenzverdopplung (SHG) und kohärente anti-Stokes Raman-Streuung (CARS), können Morphologie, chemische Zusammensetzung sowie krankheitsbedingte Veränderungen komplexer Gewebe label-frei analog zu histologischen Färbungen dargestellt werden. Potentiell eignet sich die Methode daher für die in vivo Bildgebung und könnte die medizinische Diagnostik entscheidend verbessern. Im Rahmen dieser Arbeit wurde ein kompaktes TPEF/SHG/CARS-Forschungsmikroskop unter Verwendung neuer Faserlaserquellen speziell für die Verwendung in der Klinik entwickelt. Dabei wurde erforscht, wie sich der Bildkontrast durch nahinfrarote Laser sowie eine hohe spektrale Auflösung verbessern lässt. Zusätzlich wurde an Methoden der Datenanalyse multispektraler CARS-Daten gearbeitet, um mittels der Kolokalisationsanalyse die Verteilung verschiedener molekularer Marker in komplexen Geweben zu visualisieren. Das Potential für klinische Anwendungen wurde an verschiedenen Krankheitsbildern wie Arteriosklerose und Tumoren des Hirns demonstriert

Emerging applications in mass spectrometry imaging; enablers and roadblocks

Mass spectrometry imaging (MSI) is a powerful and versatile technique able to investigate the spatial distribution of multiple non-labelled endogenous and exogenous analytes simultaneously, within a wide range of samples. Over the last two decades, MSI has found widespread application for an extensive range of disciplines including pre-clinical drug discovery, clinical applications and human identification for forensic purposes. Technical advances in both instrumentation and software capabilities have led to a continual increase in the interest in MSI; however, there are still some limitations. In this review, we discuss the emerging applications in MSI that significantly impact three key areas of mass spectrometry (MS) research—clinical, pre-clinical and forensics—and roadblocks to the expansion of use of MSI in these areas

High-Resolution Label Free Imaging of Endogenous Chromophore via Non-Linear Photoacoustic Microscopy

Molecular specific subcellular imaging of biological tissues is vital for understanding the mechanisms of various pathologies. Current technologies for subcellular absorption contrast imaging, such as fluorescence confocal microscopy, require exogenous contrast agents to gain access to relevant biomolecules. All non-fluorescing biomolecules must therefore be tagged by a fluorescent marker to be visible in fluorescence confocal images. While these markers are effective, they can change the local environments, and any exogenous contrast agent must first achieve FDA approval for wide-spread use in humans. Photoacoustic microscopy (PAM) is a hybrid imaging modality combining optical absorption imaging with ultrasonic detection capable of endogenous absorption contrast. Unfortunately, traditional photoacoustic microscopy suffers from poor axial resolution, precluding it from three-dimensional subcellular imaging. High axial resolution may be lent to PAM through the addition of a pump-probe spectroscopy technique known as transient absorption. This high resolution PAM technique, known as transient absorption ultrasonic microscopy (TAUM) enables three-dimensional subcellular imaging of endogenous biomolecules. The pump-probe spectroscopy properties inherent to TAUM provide optically resolved point spread functions, access to ground state recovery time, and access to transient absorption spectrum measurements. This manuscript describes the author’s efforts to improve the processing capabilities of both PAM and TAUM. In this manuscript various TAUM systems are designed and characterized in detail. A second generation TAUM system improves the processing speed of TAUM to enable processing in parallel with data acquisition. Following the improvements to processing, a novel optical schematic of TAUM is developed, greatly simplifying the design requirements of TAUM images. This system is validated by collecting volumetric images of erythrocytes in blood smears. This work enables any PAM system to be converted to a TAUM system through the addition of an optical modulator. The culmination of this work is a multispectral TAUM system hybridized with a confocal microscope to enable high resolution imaging with both scattering and absorption contrast of biological tissues. The capabilities of this PAM and TAUM are demonstrated by obtaining high resolution images of the endogenous chromophores: hemoglobin, melanin, and cytochrome C

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

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

Développement d’un cathéter multimodal visant l’étude de la plaque d’athérosclérose

Résumé L’objectif de cette thèse est de concevoir et valider un système d’imagerie par cathéter visant l’étude de la plaque d’athérosclérose. L’innovation repose dans l’intégration de plusieurs modalités d’imagerie dans un seul appareil. En effet, le système combine des techniques d’imagerie anatomique et moléculaire. Ceci permet d’obtenir de l’information riche et diversifiée en temps réel à propos de la plaque d’athérosclérose. Le système conçu permet d’effectuer simultanément l’imagerie ultrasonore intravasculaire (IVUS), l’imagerie photoacoustique intravasculaire (IVPA), ainsi que l’imagerie de fluorescence intravasculaire (NIRF). L’élastographie intravasculaire (IVE) est également possible en post-traitement. L’hypothèse de travail est que la combinaison de l’ensemble de ces techniques d’imagerie permet une étude plus détaillée que l’utilisation d’une seule modalité. L’IVUS est une technique d’imagerie morphologique largement utilisée en recherche clinique qui permet d’obtenir en temps réel des séries de coupes transversales des artères à haute résolution. La paroi artérielle peut être étudiée afin d’identifier certains types de plaque. L’IVUS est la composante principale du système d’imagerie par cathéter conçu dans le cadre de ce projet. Bien que cette technique d’imagerie permette de visualiser l’anatomie générale de l’artère, elle permet difficilement de caractériser les composantes principales d’une plaque vulnérable. Afin de complémenter l’IVUS, des techniques d’imagerie moléculaire, l’IVPA et la NIRF, ont été incorporées au système. Le but est d’étudier le développement de la plaque à un stade d’évolution plus précoce, alors qu’il y a inflammation de la paroi artérielle, mais une accumulation insuffisante de dépôts lipidiques pour être visibles en IVUS. L’imagerie moléculaire a le potentiel de mieux caractériser les plaques vulnérables, qui sont plus susceptibles de subir des complications, telle une thrombose. Une des applications potentielles est l’étude de l’effet de nouveaux médicaments, qui ne se traduit pas nécessairement par un changement anatomique perceptible en IVUS, mais plutôt par un changement au niveau moléculaire. L’IVE permet d’obtenir de l’information quantitative à propos des propriétés élastiques de la paroi artérielle. Elle vise à complémenter l’IVUS en fournissant des propriétés mécaniques de l’artère et en évaluant la vulnérabilité de la plaque d’athérosclérose. La première contribution de ce travail contient une description détaillée du système d’imagerie par cathéter qui a été conçu. Une preuve de concept est ensuite présentée en exposant des résultats sur fantômes exprimant un contraste dans toutes les modalités d’imagerie intégrées au système : l’IVUS, l’IVPA, la NIRF et l’IVE.La deuxième contribution pousse la validation du système plus loin en évaluant le potentiel de détection de la plaque d’athérosclérose in vivo chez le lapin. La combinaison de l’IVUS et de la NIRF, avec injection d’un biomarqueur, soit le vert d’indocyanine (ICG), a permis la détection de certaines plaques et a été comparée avec des techniques d’imagerie ex vivo. La performance ainsi que la reproductibilité des mesures ont été évaluées. La troisième contribution est reliée à la colocalisation des images en IVUS et en NIRF obtenues chez le lapin avec des images ex vivo volumétriques à très haute résolution. Les images ex vivo sont comparées à celles obtenues avec le cathéter, afin de mieux apprécier les capacités et les limites du système d’imagerie in vivo conçu.----------Abstract The aim of this thesis is to design and validate a catheter imaging system for the study of the atherosclerotic plaque. The innovation resides in the integration of multiple imaging modalities in a single device. Indeed, the system combines anatomical and molecular imaging techniques. This allows obtaining rich and diversified information in real time about the atherosclerotic plaque.The designed system allows simultaneously performing intravascular ultrasound imaging (IVUS), intravascular photoacoustic imaging (IVPA) and intravascular fluorescence (NIRF). Intravascular elastography (IVE) is also possible in post-processing. The hypothesis of this work is that the combination of all these imaging techniques allows a more detailed study than the usage of a single modality.IVUS is a morphological imaging technique widely used in preclinical research that allows obtaining in real time series of cross sections of arteries at a high resolution. The artery wall can be studied to identify certain types of plaque. IVUS is the main component of the catheter imaging system designed in this project. While this imaging technique allows visualizing the general anatomy of the artery, it is not well suited for characterizing the main components of a vulnerable plaque. To complement IVUS, molecular imaging techniques, IVPA and NIRF, were incorporated to the system. The goal is to study the development of the plaque at an earlier evolution stage when there is inflammation in the artery wall, but an insufficient accumulation of lipids to be visible in IVUS. Molecular imaging has the potential to better characterize vulnerable plaques, which are more prone to complications, such as thrombosis. One of the potential applications is the study of the effect of new drugs, that doesn’t always translate by an anatomical change perceptible in IVUS, but rather a change at the molecular level. IVE allows obtaining quantitative information about elastic properties of the artery wall. It aims at complementing IVUS by providing mechanical properties of the artery and by evaluating the vulnerability of atherosclerotic plaque. The first contribution to this work contains a detailed description of the designed catheter imaging system. A proof of concept is then presented by exposing results on phantoms with contrasts in all the imaging modalities integrated to the system: IVUS, IVPA, NIRF and IVE. The second contribution further validates the system by evaluating the detection potential of atherosclerotic plaque in vivo on rabbits. The IVUS and NIRF combination, with the injection of a biomarker, indocyanine green (ICG), allowed the detection of certain plaques and was compared to ex vivo imaging techniques. The performance and the reproducibility of the measures were evaluated. The third contribution is related to the colocalisation of IVUS and NIRF images obtained in rabbits with volumetric ex vivo images at a very high resolution. Ex vivo images are compared to the ones obtained with the catheter, in order to better appreciate the capabilities and the limitations of the designed in vivo imaging system

Raman Spectroscopy in Nanomedicine: Current Status and Future Perspectives

Raman spectroscopy is a branch of vibration spectroscopy which is capable of probing the chemical composition of materials. Recent advances in Raman microscopy have added significantly to the range of applications which now extend from medical diagnostics to exploring interfaces between biological organisms and nanomaterials. In this review, Raman is introduced in a general context, highlighting some of the areas in which the technique has found success in the past, as well as some of the potential benefits it offers over other analytical modalities. The subset of Raman techniques which specifically probe the nanoscale, namely Surface Enhanced and Tip Enhanced Raman Spectroscopy, will be described and specific applications relevant to nanomedical applications will be reviewed. Progress in the use of traditional label-free Raman applied to investigation of nanoscale interactions will be described, and recent developments in Coherent Anti-Stokes Raman Scattering will be explored, particularly applications to biomedical and nanomedical fields