Development and characterization of a microscope based on pump-probe spectroscopy: a valuable tool for the study of photoactivated drugs in cellulo

This thesis presents the construction and characterization of a microscope based on pumpprobe spectroscopy and its application to observe and study the evolution of photoinduced reactions. As compared to the typical pump-probe systems reported in the literature, which utilizes either 1 kHz or 80 MHz pulse-trains, the developed microscope was fed by pump pulse-trains at 125 kHz and probe pulse-trains at 250 kHz repetition rates in order to perform transient absorption microscopy (TAM). The use of such intermediate repetition rate reduces the unnecessary exposure of the sample to input field leading to risk of photo-induced damage and it also relaxes the use of lock-in detection, an unavoidable element for any MHz system, which increases the cost and complexity of the system. This gives the possibility to perform microspectroscopic studies of micro-structures with minimal photo-induced damage, a property that is especially important to the study of biological samples, without the incorporation of lock-in detection. Therefore, the developed TAM system by itself is a proof of concept.In der vorliegenden Arbeit wurde die Konstruktion, Wirkungsweise und Anwendung eines mikroskopgestützten Pump-Probe Spektroskops Mikroskops sowie dessen Einsatz zu Lokalisierung und Beobachtung photoneninduzierter Reaktionen beschrieben. Sie soll sowohl als Einführung in die Pump-Probe Mikroskopie dienen als auch die Möglichkeiten dieser Technik herausstellen, welche Wissenschaftlern neue Sichtweisen auf ultraschnelle photonenindizierte Prozesse sowie Informationen zur Verteilung von medizinischen Wirkstoffen innerhalb einer Zelle liefern könnten. Dies kann ein Hinweis darauf liefern, wie sich ein untersuchtes Medikament in der Zelle bzw. des jeweiligen Zellbestandteils verhält. Ein solches mikrospektroskopisches Werkzeug ermöglicht nun die Charakterisierung von ausgewählten Molekülen in cellulo bezüglich ihrer photo-physikalischen Eigenschaften. Es wurde gezeigt, dass durch die spezifischen Eigenschaften der Pump-Probe Mikroskopie die Möglichkeit besteht die zeitliche Entwicklung von photoaktivierter Stoffe mit beugungsbegrenzter Auflösung zu untersuchen

Diseño e implementación de un banco de pruebas de inyectores de vehículos a diesel CRDI para la Escuela de Ingeniería Automotriz.

Este trabajo trata sobre el diseño e implementación de un banco de pruebas de inyectores de vehículos a diesel CRDI, con el objetivo de diagnosticar el funcionamiento o no de los inyectores de todos los vehículos de combustible diésel. En el país se ha ido incrementando en gran cantidad los vehículos con sistemas de inyección diesel como son: BOSCH, DELPHI, DENSO, siendo inapropiado el diagnostico de funcionamiento y análisis de averías de los inyectores. Este proyecto implementa el sistema de módulo controlador de pulsos electromagnéticos, el mismo que hace el trabajo de una ECU en un vehículo diésel, es decir da el mando para que el inyector a través de los pulsos, inyecte el combustible, abriendo y cerrando la tobera del inyector, con los datos establecidos en el módulo controlador. Además para que el inyector cumpla su función de inyección se incorpora un manómetro que hace la función de bomba para enviar presión al inyector y así poder comprobar si está cumpliendo o no con su funcionamiento normal y adecuado. Los estudios de los sistema de inyección directa en los vehículos a diesel, lleva a la invención, diseño, estudio técnico para la elaboración de un sistema de banco de pruebas, que genera pulsos electromagnéticos para controlar los inyectores (pulsos: 200, DUTY: ralentí: 40% y plena carga: 80%), implementando un diagnóstico más técnico, a menor tiempo y costo. Los resultados obtenidos son los siguientes: si los niveles de entrega y retorno son iguales: el inyector funciona regularmente pero debe ser reparado; si el nivel de entrega es mayor al nivel de retorno: el inyector funciona normalmente; si el nivel de retorno es mayor que el nivel de entrega hay una fuga y el vehículo no enciende; se recomienda antes de realizar un análisis de funcionamiento, utilizar equipos de seguridad y protección; además los inyectores deben ser reparados para funcionar correctamente, pero no necesariamente pueden solucionar el problema. Para un rendimiento óptimo y eficaz, todos los inyectores deben fluir a la misma cantidad de combustible y tener una buena atomización de combustible.This investigation is about the design and implementation of a test injector for diesel powered vehicles CRDI (Common Rail Diesel Injection), in order to diagnose if the injectors of all diesel powered vehicles work or not in a correct way. Vehicles with a diesel system have been increasing in a large number in the country; there are: BOSCH, DELPHI, and DENSO, but the operation diagnosis and injectors breakdown analysis are inappropriate. This project implements the electromagnetic pulse module controller system, which works as an ECU (Engine Control Unit) in a diesel vehicle; it means that the injector injects fuel by means of pulses, in this way it opens and closes the injector nozzle, with data established in the module controller, moreover, a pressure gauge is also implemented in order to fulfill the injection function which works a pump to send pressure to the injector, so this way you can check fi it fulfills or not with its normal and adequate operation. The surveys on direct injection system for diesel powered vehicles allow the creation, design, and technical research for the development of a test injector, which generates electromagnetic pulses to control the injector´s duty slow motion 40% and full charge 80%. This allowed implementing a more technical diagnosis with less time and cost. The results obtained are the following: if the fuel intake level and return level are similar: the injector works regularly, but it must be repaired, if the intake level is higher than the return level the injector works normally, if the return level is higher than the intake level, there is a leakage and the engine doesn´t start. Before making and operating analysis, it is recommended to use and wear safety and protection equipment and the injectors must also be repaired for a good operating, but they don´t necessarily solve the problem. For having an optimal and efficient performance, it is necessary that all the injectors have the same fuel flow to obtain a correct fuel atomization

Ultrafast in cellulo photoinduced dynamics processes of the paradigm molecular light switch [Ru(bpy)2dppz]2+

An in cellulo study of the ultrafast excited state processes in the paradigm molecular light switch [Ru(bpy)2dppz]2+ by localized pump-probe spectroscopy is reported for the first time. The localization of [Ru(bpy)2dppz]2+ in HepG2 cells is verified by emission microscopy and the characteristic photoinduced picosecond dynamics of the molecular light switch is observed in cellulo. The observation of the typical phosphorescence stemming from a 3MLCT state suggests that the [Ru(bpy)2dppz]2+ complex intercalates with the DNA in the nucleus. The results presented for this benchmark coordination compound reveal the necessity to study the photoinduced processes in coordination compounds for intracellular use, e.g. as sensors or as photodrugs, in the actual biological target environment in order to derive a detailed molecular mechanistic understanding of the excited-state properties of the systems in the actual biological target environment

Fingerprint multiplex CARS at high speed based on supercontinuum generation in bulk media and deep learning spectral denoising

We introduce a broadband coherent anti-Stokes Raman scattering (CARS) microscope based on a 2-MHz repetition rate ytterbium laser generating 1035-nm high-energy (≈µJ level) femtosecond pulses. These features of the driving laser allow producing broadband red-shifted Stokes pulses, covering the whole fingerprint region (400-1800 cm-1), employing supercontinuum generation in a bulk crystal. Our system reaches state-of-the-art acquisition speed (<1 ms/pixel) and unprecedented sensitivity of ≈14.1 mmol/L when detecting dimethyl sulfoxide in water. To further improve the performance of the system and to enhance the signal-to-noise ratio of the CARS spectra, we designed a convolutional neural network for spectral denoising, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues

Invited Article: Complex vibrational susceptibility by interferometric Fourier transform stimulated Raman scattering

We introduce interferometric (I) Fourier-transform (FT) stimulated Raman scattering (SRS) to measure the complex nonlinear vibrational susceptibility of molecules. The technique is a simple variation of FT-SRS, which was previously demonstrated to combine the very high sensitivity of single-channel lock-in detection with the spectral resolution afforded by FT spectroscopy. In IFT-SRS, a local oscillator, temporally anticipated with respect to the broadband pump pulse, enables the interferometric detection of both real and imaginary parts of the nonlinear susceptibility, whose spectrum is recorded in the time domain by scanning the delay of the local oscillator using a birefringent common-path interferometer. We apply IFT-SRS to record the complex vibrational response of different solvents and their mixtures. (C) 2018 Author(s)

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections

Broadband stimulated Raman imaging based on multi-channel lock-in detection for spectral histopathology

Spontaneous Raman microscopy reveals the chemical composition of a sample in a label-free and non-invasive fashion by directly measuring the vibrational spectra of molecules. However, its extremely low cross section prevents its application to fast imaging. Stimulated Raman scattering (SRS) amplifies the signal by several orders of magnitude thanks to the coherent nature of the nonlinear process, thus unlocking high-speed microscopy applications that provide analytical information to elucidate biochemical mechanisms with subcellular resolution. Nevertheless, in its standard implementation, narrowband SRS provides images at only one frequency at a time, which is not sufficient to distinguish constituents with overlapping Raman bands. Here, we report a broadband SRS microscope equipped with a home-built multichannel lock-in amplifier simultaneously measuring the SRS signal at 32 frequencies with integration time down to 44 μs, allowing for detailed, high spatial resolution mapping of spectrally congested samples. We demonstrate the capability of our microscope to differentiate the chemical constituents of heterogeneous samples by measuring the relative concentrations of different fatty acids in cultured hepatocytes at the single lipid droplet level and by differentiating tumor from peritumoral tissue in a preclinical mouse model of fibrosarcoma

Factors associated with cognitive impairment in Latin American older adults: A cross‐sectional observational study of COVID‐19 confinement

INTRODUCTION: The effects of COVID-19 confinement have been severe, especially in older adults. Therefore, we analyzed the factors associated with cognitive impairment (CI) in Latin America (LA). METHODS: We conducted a cross-sectional observational study with a total of 5245 older adults from 10 countries in LA. Measurement: We used the Telephone Montreal Cognitive Assessment (T-MoCA) and the Eight-item Informant Interview to Differentiate Aging and Dementia (AD8) scale. RESULTS: We found that age, depressive symptomatology, bone fractures, being widowed, having a family member with dementia, and unemployment were associated with an increased risk of CI. In contrast, higher education, hypertension with continuous treatment, quarantine, and keeping stimulating cognitive and physical activities were associated with a lower probability of CI. No significant association was found between suffering from diabetes or being retired and CI. DISCUSSION: It is essential to conduct follow-up studies on these factors, considering their relationship with CI and the duration of confinement

Ontologías en disputa: Diálogos entre la antropología y la arqueología para la problematización de paisajes regionals

Objetivo/contexto: Hacia finales del siglo XX, el dualismo ontológico con el que antropólogos y arqueólogos nos hemos formado teórica y metodológicamente ha sido cuestionado y puesto en debate. En este contexto, nuestro objetivo es exponer críticamente los trabajos que se están realizando y que contribuyen a problematizar los paisajes enredados de existencias, materialidades, saberes, sentidos o prejuicios pasados y presentes, y con ello a restablecer el diálogo entre la antropología social y la arqueología. Metodología: Exponemos el estado actual de la apertura ontológica en clave latinoamericana. Problematizamos la noción de paisaje como concepto que permite diálogos teórico-metodológicos entre la antropología y la arqueología, así como con otras disciplinas. También presentamos ejes de comunicación y diálogo entre los artículos que forman parte del presente dossier para finalizar con los desafíos por delante en cuanto a traspasar fronteras disciplinarias, temporales y espaciales. Conclusiones: En contextos de históricas relaciones de poder, de profundas crisis socio-ambientales, los principales desafíos de la apertura ontológica serían: 1) generar referentes teóricos permeables a diversas disciplinas y saberes; 2) consolidar metodologías que integren lo etnográfico con lo arqueológico; 3) alentar diálogos constructivos hacia y con otras disciplinas y saberes, y 4) propiciar la comprensión del entramado político actual y la generación de políticas públicas que permitan diversas formas de relacionamiento con el entorno. Originalidad: El restablecimiento de diálogos entre la arqueología y la antropología social desde las aperturas ontológicas tiene el potencial de discutir cómo en determinados contextos se concretan y/o disputan paisajes y modos hegemónicos de habitar en el mundo, así como se problematizan los usos políticos del pasado y las políticas de uso del espacio y el ambiente.Objective/context: Towards the end of the 20th century, the ontological dualism with which anthropologists and archaeologists have trained us theoretically and methodologically has been questioned and debated. In this context, our objective is to critically expose the works that are being carried out and that contribute to problematizing the landscapes entangled with past and present existences, materialities, knowledge, senses or prejudices, and, with it, to re-establish the dialogue between social anthropology and archaeology. Methodology: We expose the current state of the ontological opening in Latin American code. We problematize the notion of landscape as a concept that allows theoretical-methodological dialogues between anthropology and archaeology, as well as with other disciplines. We also present axes of communication and dialogue between the articles that are part of this dossier, to end with the challenges ahead in terms of crossing disciplinary, temporal and spatial borders. Conclusions: In contexts of historical power relations, of deep socio-environmental crises, the main challenges for an ontological opening would be: 1) to generate theoretical referents permeable to various disciplines and fields of knowledge; 2) to consolidate methodologies that integrate the ethnographic with the archaeological; 3) to encourage constructive dialogues towards and with other disciplines and fields of knowledge; and 4) to propitiate the understanding of the current political framework and the generation of public policies that allow for diverse forms of relation with the environment. Originality: The re-establishment of dialogues between archaeology and social anthropology from ontological openings has the potential to discuss how, in certain contexts, landscapes and hegemonic ways of living are concretized and/or disputed, as well as the political uses of the past and the policies of use of space and the environment.Objetivo/contexto: No final do século XX, o dualismo ontológico com o qual antropólogos e arqueólogos se formaram teórica e metodologicamente vem sendo questionado e colocado em debate. Nesse contexto, nosso objetivo é expor criticamente os trabalhos que estão sendo realizados e que contribuem para problematizar as paisagens permeadas de existências, materialidades, saberes, sentidos ou preconceitos passados e presentes, e, com isso, reestabelecer o diálogo entre a antropologia social e a arqueologia. Metodologia: Expomos o estado atual da abertura ontológica no âmbito latino-americano. Problematizamos a noção de paisagem como conceito que permite diálogos teórico-metodológicos entre a antropologia e a arqueologia, assim como outras disciplinas. Também apresentamos eixos de comunicação e diálogo entre os artigos que fazem parte do presente dossiê para finalizar com os desafios que temos adiante sobre ultrapassar fronteiras disciplinares, temporais e espaciais. Conclusões: Em contextos de históricas relações de poder, de profundas crises socioambientais, os principais desafios para uma abertura ontológica seriam: 1) gerar referentes teóricos permeáveis a diversas disciplinas e saberes; 2) consolidar metodologias que integrem o etnográfico com o arqueológico; 3) promover diálogos construtivos com outras disciplinas e saberes; e 4) propiciar a compreensão da estrutura política atual e a geração de políticas públicas que permitam as diversas formas de relacionamentos com o ambiente. Originalidade: O reestabelecimento de diálogos entre a arqueologia e a antropologia social a partir das aberturas ontológicas tem o potencial de discutir como, em determinados contextos, se concretizam e/ou disputam paisagens e modos hegemônicos de habitar, bem como se problematizam os usos políticos do passado e as políticas de uso do espaço e do ambiente.Fil: Saldi, Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Mafferra, Luis Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Barrientos Salinas, J. Alejandro. Universidad Mayor de San Andrés; Bolivi

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection