Nanophotonic antennas for enhanced single-molecule fluorescence detection and nanospectroscopy in living cells membranes

Single-molecule fluorescence spectroscopy has revolutionized the field of biophysical sciences by enabling visualization of dynamic molecular interactions and nanoscopic features with high spatiotemporal resolution. Monitoring enzymatic reactions and studying diffusion dynamics of individual molecules (such as lipids and proteins) help us understand how these nanoscopic entities influence and control various biochemical processes. Nanophotonic antennas can efficiently localize electromagnetic radiation into nanoscale spatial dimensions comparable to single bio-molecules (<10 nm). These ultra-confined illumination hotspots thereby offer opportunity to follow single-molecule events at physiological expression levels. In this thesis, we explore various photonic nanoantenna platforms (double nanohole apertures, dimer nanogap antennas and planar "antenna-in-box'') and demonstrate their application in enhanced single-molecule fluorescence detection. Using fluorescence burst analysis, fluorescence correlation spectroscopy (FCS), time-correlated TCSPC measurements, and near field simulations, we quantify nanoantenna detection volumes, fluorescence enhancement factors and discuss the fluorescence photodynamic accelerations mediated by optical nanoantennas. An alternative to plasmonic structures, all-dielectric nanoantenna based on silicon nanogap is also demonstrated to enhance the fluorescence detection of single molecules diffusing in concentrated solutions. Further, using resonant planar "antenna-in-box'' devices we investigate the diffusion dynamics of phosphoethanolamine and sphingomyelin on the plasma membrane of living cells and discuss the results in the context of lipid rafts. Together with cholesterol depletion experiments, we provide evidence of cholesterol-induced nanodomain partitioning within less than 10 nm diameters and characteristic times being ~100 microsecondsLa espectroscopia de fluorescencia de una sola molecula ha revolucionado el campo de las ciencias biofisicas, permitiendo la visualizacion de interacciones moleculares dinamicas y caracteristicas nanoscopicas con alta resolucion espaciotemporal. La monitorizacion de las reacciones enzimaticas y el analisis de la dinamica de difusion de moleculas individuales (como lipidos y proteinas) nos ayudan a comprender como estas entidades nanoscopicas influyen y controlan diversos procesos bioquimicos. Las antenas nanofotonicas pueden localizar eficientemente la radiacion electromagnetica en dimensiones espaciales en nanoescala, comparables a biomoleculas unicas (<10 nm). Estos hotspots de iluminacion ultra configurados ofrecen de este modo la oportunidad de monitorizar eventos de molecula unica a niveles de expresion fisiologica. En esta tesis, exploramos varias plataformas fotonicas de nanoantenas (double nanohole aperture, dimero nanogap antenas y "antenna-in-box" planares) y demostramos su aplicacion en la mejora de la deteccion una sola molecula de fluorescencia. Utilizando el analisis por explosion de fluorescencia, espectroscopia de correlacion de fluorescencia (FCS), medidas TCSPC correlacionadas en el tiempo y simulaciones de campo cercano, cuantificamos volumenes de deteccion de nanoantenas, factores de mejora de fluorescencia y discutimos las aceleraciones fotodinámicas de fluorescencia mediada por nanoantennas opticas. Las nanoantennas dielectricas basadas en nanogaps de silico se han propuesto como una alternativa en el realce de la deteccion de fluorescencia de difusion de moleculas unicas en soluciones concentradas. Ademas, utilizando dispositivos resonantes planares de "antenna-in-box", investigamos la dinamica de difusion de la fosfoetanolamina y la esfingomielina en la membrana plasmatica de las celulas vivas y discutimos los resultados en el contexto de las balsas lipidicas. Junto con experimentos de dismincion de colesterol, proporcionamos pruebas de division inducida por colesterol en el nanodominio dentro de diametros menors de 10 nm y con tiempos caracteristicos de ~100 microsegundos.La spectroscopie de fluorescence d'une seule molécule a révolutionné le domaine des sciences biophysiques, permettant la visualisation d'interactions moléculaires dynamiques et de caractéristiques nanoscopiques à haute résolution spatio-temporelle. Le suivi des réactions enzymatiques et l'analyse de la dynamique de diffusion des molécules individuelles (telles que les lipides et les protéines) nous aident à comprendre comment ces entités nanoscopiques influencent et contrôlent divers processus biochimiques. Les antennes nanophotoniques peuvent localiser efficacement le rayonnement électromagnétique à des dimensions spatiales nanométriques, comparables à des biomolécules uniques (<10 nm). Ces hotspots d'éclairage ultra-configurés offrent la possibilité de surveiller les événements de molécules uniques à des niveaux d'expression physiologiques. Dans ce mémoire, nous examinons plusieurs plates-formes photoniques nanoantennas (nanotrou à double ouverture, I antennes Dimer nanoespace et plane « antenne-in-box ») et de démontrer son application dans l'amélioration de la détection d'une fluorescence seule molécule. Utilisation de l'analyse par spectroscopie de fluorescence d'explosion corrélation de fluorescence (FCS), les mesures TCSPC corrélées dans le temps et proches des simulations champ quantifier les volumes de détection de nanoantennas, les facteurs d'amélioration fluorescence et discuter des accélérations photodynamiques fluorescence médiée nanoantennas opticas. Des nanoantennas diélectriques à base de nanogap silico ont été proposées comme alternative dans l'amélioration de la détection par fluorescence de la diffusion de molécules uniques dans des solutions concentrées. En outre, l'utilisation de "plan d'antenne-in-box" dispositifs de résonance, nous étudions la dynamique de diffusion de phosphoéthanolamine et sphingomyéline dans la membrane plasmique des cellules vivantes et de discuter des résultats dans le contexte des radeaux lipidiques. Conjointement avec des expériences de réduction du cholestérol, nous fournissons des tests de division induits par le cholestérol dans le nanodomaine dans des diamètres plus petits de 10 nm et avec des temps caractéristiques de ~ 100 microsecondes

Nanophotonic antennas for enhanced single-molecule fluorescence detection and nanospectroscopy in living cells membranes

Cotutela Universitat Politècnica de Catalunya i Aix-Marseille UniversitéSingle-molecule fluorescence spectroscopy has revolutionized the field of biophysical sciences by enabling visualization of dynamic molecular interactions and nanoscopic features with high spatiotemporal resolution. Monitoring enzymatic reactions and studying diffusion dynamics of individual molecules (such as lipids and proteins) help us understand how these nanoscopic entities influence and control various biochemical processes. Nanophotonic antennas can efficiently localize electromagnetic radiation into nanoscale spatial dimensions comparable to single bio-molecules (<10 nm). These ultra-confined illumination hotspots thereby offer opportunity to follow single-molecule events at physiological expression levels. In this thesis, we explore various photonic nanoantenna platforms (double nanohole apertures, dimer nanogap antennas and planar "antenna-in-box'') and demonstrate their application in enhanced single-molecule fluorescence detection. Using fluorescence burst analysis, fluorescence correlation spectroscopy (FCS), time-correlated TCSPC measurements, and near field simulations, we quantify nanoantenna detection volumes, fluorescence enhancement factors and discuss the fluorescence photodynamic accelerations mediated by optical nanoantennas. An alternative to plasmonic structures, all-dielectric nanoantenna based on silicon nanogap is also demonstrated to enhance the fluorescence detection of single molecules diffusing in concentrated solutions. Further, using resonant planar "antenna-in-box'' devices we investigate the diffusion dynamics of phosphoethanolamine and sphingomyelin on the plasma membrane of living cells and discuss the results in the context of lipid rafts. Together with cholesterol depletion experiments, we provide evidence of cholesterol-induced nanodomain partitioning within less than 10 nm diameters and characteristic times being ~100 microsecondsLa espectroscopia de fluorescencia de una sola molecula ha revolucionado el campo de las ciencias biofisicas, permitiendo la visualizacion de interacciones moleculares dinamicas y caracteristicas nanoscopicas con alta resolucion espaciotemporal. La monitorizacion de las reacciones enzimaticas y el analisis de la dinamica de difusion de moleculas individuales (como lipidos y proteinas) nos ayudan a comprender como estas entidades nanoscopicas influyen y controlan diversos procesos bioquimicos. Las antenas nanofotonicas pueden localizar eficientemente la radiacion electromagnetica en dimensiones espaciales en nanoescala, comparables a biomoleculas unicas (<10 nm). Estos hotspots de iluminacion ultra configurados ofrecen de este modo la oportunidad de monitorizar eventos de molecula unica a niveles de expresion fisiologica. En esta tesis, exploramos varias plataformas fotonicas de nanoantenas (double nanohole aperture, dimero nanogap antenas y "antenna-in-box" planares) y demostramos su aplicacion en la mejora de la deteccion una sola molecula de fluorescencia. Utilizando el analisis por explosion de fluorescencia, espectroscopia de correlacion de fluorescencia (FCS), medidas TCSPC correlacionadas en el tiempo y simulaciones de campo cercano, cuantificamos volumenes de deteccion de nanoantenas, factores de mejora de fluorescencia y discutimos las aceleraciones fotodinámicas de fluorescencia mediada por nanoantennas opticas. Las nanoantennas dielectricas basadas en nanogaps de silico se han propuesto como una alternativa en el realce de la deteccion de fluorescencia de difusion de moleculas unicas en soluciones concentradas. Ademas, utilizando dispositivos resonantes planares de "antenna-in-box", investigamos la dinamica de difusion de la fosfoetanolamina y la esfingomielina en la membrana plasmatica de las celulas vivas y discutimos los resultados en el contexto de las balsas lipidicas. Junto con experimentos de dismincion de colesterol, proporcionamos pruebas de division inducida por colesterol en el nanodominio dentro de diametros menors de 10 nm y con tiempos caracteristicos de ~100 microsegundos.La spectroscopie de fluorescence d'une seule molécule a révolutionné le domaine des sciences biophysiques, permettant la visualisation d'interactions moléculaires dynamiques et de caractéristiques nanoscopiques à haute résolution spatio-temporelle. Le suivi des réactions enzymatiques et l'analyse de la dynamique de diffusion des molécules individuelles (telles que les lipides et les protéines) nous aident à comprendre comment ces entités nanoscopiques influencent et contrôlent divers processus biochimiques. Les antennes nanophotoniques peuvent localiser efficacement le rayonnement électromagnétique à des dimensions spatiales nanométriques, comparables à des biomolécules uniques (<10 nm). Ces hotspots d'éclairage ultra-configurés offrent la possibilité de surveiller les événements de molécules uniques à des niveaux d'expression physiologiques. Dans ce mémoire, nous examinons plusieurs plates-formes photoniques nanoantennas (nanotrou à double ouverture, I antennes Dimer nanoespace et plane « antenne-in-box ») et de démontrer son application dans l'amélioration de la détection d'une fluorescence seule molécule. Utilisation de l'analyse par spectroscopie de fluorescence d'explosion corrélation de fluorescence (FCS), les mesures TCSPC corrélées dans le temps et proches des simulations champ quantifier les volumes de détection de nanoantennas, les facteurs d'amélioration fluorescence et discuter des accélérations photodynamiques fluorescence médiée nanoantennas opticas. Des nanoantennas diélectriques à base de nanogap silico ont été proposées comme alternative dans l'amélioration de la détection par fluorescence de la diffusion de molécules uniques dans des solutions concentrées. En outre, l'utilisation de "plan d'antenne-in-box" dispositifs de résonance, nous étudions la dynamique de diffusion de phosphoéthanolamine et sphingomyéline dans la membrane plasmique des cellules vivantes et de discuter des résultats dans le contexte des radeaux lipidiques. Conjointement avec des expériences de réduction du cholestérol, nous fournissons des tests de division induits par le cholestérol dans le nanodomaine dans des diamètres plus petits de 10 nm et avec des temps caractéristiques de ~ 100 microsecondes.Postprint (published version

Prevalence of Helicobacter Pylori among Patients undergoing Gastrodudenoscopy in a Hospital in Western Nepal

Introduction: Helicobacter pylori (H. pylori) related chronic gastritis is a major health problem worldwide, specially in the developing countries. The prevalence of H. pylori infection has been reported to vary between and even within countries. There are limited data on this infection in Western Nepal. Our objective was to study the prevalence of H. pylori infection and its association with presenting complains and upper gastrointestinal diseases. Methods: Medical records of patients undergoing gastrodudenoscopy and biopsy for various upper gastrointestinal symptoms from 1st of January 2015 to 30th of June 2017 were reviewed for presence of H. pylori infection, demographics, indications for gastrodudenoscopy, and histopathology findings. T-test, Chi-square test, and Fisher exact test were applied. Results: Two hundred fifty six patients (135 male and 121 female) with a mean age of 47 (SD = 16.5) underwent gastroscopic biopsy and had an overall H. pylori prevalence of 24.6%. H. pylori infection was most commonly noted between 41 to 60 years of age. Gender did not seem to be significantly associated (p = 0.82) but gastrointestinal bleed was significantly associated with H. pylori infection (p = 0.006). The most common histopathological diagnosis was gastritis followed by gastrodudenitis; however, none of the diagnosis were found to be significantly associated with H. pylori infection. Conclusion: The overall prevalence of H. pylori infection was 24.6% and was most common between 40 to 60 years of age. Heart burn was the most common symptom and gastrointestinal bleed was the only significantly associated symptom with H. pylori infection

Nanoscale volume confinement and fluorescence enhancement with double nanohole aperture

International audienceDiffraction ultimately limits the fluorescence collected from a single molecule, and sets an upper limit to the maximum concentration to isolate a single molecule in the detection volume. To overcome these limitations, we introduce here the use of a double nanohole structure with 25 nm gap, and report enhanced detection of single fluorescent molecules in concentrated solutions exceeding 20 micromolar. The nanometer gap concentrates the light into an apex volume down to 70 zeptoliter (10 −21 L), 7000-fold below the diffraction-limited confocal volume. Using fluorescence correlation spectroscopy and time-correlated photon counting, we measure fluorescence enhancement up to 100-fold, together with local density of optical states (LDOS) enhancement of 30-fold. The distinctive features of double nanoholes combining high local field enhancement, efficient background screening and relative nanofabrication simplicity offer new strategies for real time investigation of biochemical events with single molecule resolution at high concentrations. Plasmonic nanoantennas realize a new paradigm to concentrate light energy into nanoscale dimension

A Case Report on Canine Transmissible Venereal Tumor

A male Japanese spitz (3 years) was brought at Himalayan Animal Rescue Trust (HART), Pokhara with a complaint of swollen gums and loss of appetite. A lobulated tumorous mass was seen at the gingival region on physical examination. Diagnosis and treatment of condition detected in the dog was the major objective. Impression smear of tumor cell was prepared and was observed under oil immersion microscope (100x). Microscopic examination shows the presence of vacuolations within the cytoplasm and the condition was diagnosed to be CTVT. Chemotherapy was performed using the most effective cytostatic agents I.e. Vincristine sulphate (once a week, I/v). The chemotherapy was repeated for 3 doses till the tumor gets completely regressed. The condition was resolved after third session of chemotherapy. Myelosuppression and gastrointestinal effects like vomiting are the major complications of using vincristine

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.Peer ReviewedPostprint (author's final draft

The COVID‐19 Pandemic Not Only Poses Challenges, but Also Opens Opportunities for Sustainable Transformation

The COVID-19 pandemic has impacted social, economic, and environmental systems worldwide, slowing down and reversing the progress made in achieving the Sustainable Development Goals (SDGs). SDGs belong to the 2030 Agenda to transform our world by tackling humankind's challenges to ensure well-being, economic prosperity, and environmental protection. We explore the potential impacts of the pandemic on SDGs for Nepal. We followed a knowledge co-creation process with experts from various professional backgrounds, involving five steps: online survey, online workshop, assessment of expert's opinions, review and validation, and revision and synthesis. The pandemic has negatively impacted most SDGs in the short term. Particularly, the targets of SDG 1, 4, 5, 8, 9, 10, 11, and 13 have and will continue to have weakly to moderately restricting impacts. However, a few targets of SDG 2, 3, 6, and 11 could also have weakly promoting impacts. The negative impacts have resulted from impeding factors linked to the pandemic. Many of the negative impacts may subside in the medium and long terms. The key five impeding factors are lockdowns, underemployment and unemployment, closure of institutions and facilities, diluted focus and funds for non-COVID-19-related issues, and anticipated reduction in support from development partners. The pandemic has also opened a window of opportunity for sustainable transformation, which is short-lived and narrow. These opportunities are lessons learned for planning and action, socio-economic recovery plan, use of information and communication technologies and the digital economy, reverse migration and “brain gain,” and local governments' exercising authorities

Nanophotonic antennas for enhanced single-molecule fluorescence detection and nanospectroscopy in living cells membranes

Single-molecule fluorescence spectroscopy has revolutionized the field of biophysical sciences by enabling visualization of dynamic molecular interactions and nanoscopic features with high spatiotemporal resolution. Monitoring enzymatic reactions and studying diffusion dynamics of individual molecules (such as lipids and proteins) help us understand how these nanoscopic entities influence and control various biochemical processes. Nanophotonic antennas can efficiently localize electromagnetic radiation into nanoscale spatial dimensions comparable to single bio-molecules (<10 nm). These ultra-confined illumination hotspots thereby offer opportunity to follow single-molecule events at physiological expression levels. In this thesis, we explore various photonic nanoantenna platforms (double nanohole apertures, dimer nanogap antennas and planar "antenna-in-box'') and demonstrate their application in enhanced single-molecule fluorescence detection. Using fluorescence burst analysis, fluorescence correlation spectroscopy (FCS), time-correlated TCSPC measurements, and near field simulations, we quantify nanoantenna detection volumes, fluorescence enhancement factors and discuss the fluorescence photodynamic accelerations mediated by optical nanoantennas. An alternative to plasmonic structures, all-dielectric nanoantenna based on silicon nanogap is also demonstrated to enhance the fluorescence detection of single molecules diffusing in concentrated solutions. Further, using resonant planar "antenna-in-box'' devices we investigate the diffusion dynamics of phosphoethanolamine and sphingomyelin on the plasma membrane of living cells and discuss the results in the context of lipid rafts. Together with cholesterol depletion experiments, we provide evidence of cholesterol-induced nanodomain partitioning within less than 10 nm diameters and characteristic times being ~100 microsecondsLa espectroscopia de fluorescencia de una sola molecula ha revolucionado el campo de las ciencias biofisicas, permitiendo la visualizacion de interacciones moleculares dinamicas y caracteristicas nanoscopicas con alta resolucion espaciotemporal. La monitorizacion de las reacciones enzimaticas y el analisis de la dinamica de difusion de moleculas individuales (como lipidos y proteinas) nos ayudan a comprender como estas entidades nanoscopicas influyen y controlan diversos procesos bioquimicos. Las antenas nanofotonicas pueden localizar eficientemente la radiacion electromagnetica en dimensiones espaciales en nanoescala, comparables a biomoleculas unicas (<10 nm). Estos hotspots de iluminacion ultra configurados ofrecen de este modo la oportunidad de monitorizar eventos de molecula unica a niveles de expresion fisiologica. En esta tesis, exploramos varias plataformas fotonicas de nanoantenas (double nanohole aperture, dimero nanogap antenas y "antenna-in-box" planares) y demostramos su aplicacion en la mejora de la deteccion una sola molecula de fluorescencia. Utilizando el analisis por explosion de fluorescencia, espectroscopia de correlacion de fluorescencia (FCS), medidas TCSPC correlacionadas en el tiempo y simulaciones de campo cercano, cuantificamos volumenes de deteccion de nanoantenas, factores de mejora de fluorescencia y discutimos las aceleraciones fotodinámicas de fluorescencia mediada por nanoantennas opticas. Las nanoantennas dielectricas basadas en nanogaps de silico se han propuesto como una alternativa en el realce de la deteccion de fluorescencia de difusion de moleculas unicas en soluciones concentradas. Ademas, utilizando dispositivos resonantes planares de "antenna-in-box", investigamos la dinamica de difusion de la fosfoetanolamina y la esfingomielina en la membrana plasmatica de las celulas vivas y discutimos los resultados en el contexto de las balsas lipidicas. Junto con experimentos de dismincion de colesterol, proporcionamos pruebas de division inducida por colesterol en el nanodominio dentro de diametros menors de 10 nm y con tiempos caracteristicos de ~100 microsegundos.La spectroscopie de fluorescence d'une seule molécule a révolutionné le domaine des sciences biophysiques, permettant la visualisation d'interactions moléculaires dynamiques et de caractéristiques nanoscopiques à haute résolution spatio-temporelle. Le suivi des réactions enzymatiques et l'analyse de la dynamique de diffusion des molécules individuelles (telles que les lipides et les protéines) nous aident à comprendre comment ces entités nanoscopiques influencent et contrôlent divers processus biochimiques. Les antennes nanophotoniques peuvent localiser efficacement le rayonnement électromagnétique à des dimensions spatiales nanométriques, comparables à des biomolécules uniques (<10 nm). Ces hotspots d'éclairage ultra-configurés offrent la possibilité de surveiller les événements de molécules uniques à des niveaux d'expression physiologiques. Dans ce mémoire, nous examinons plusieurs plates-formes photoniques nanoantennas (nanotrou à double ouverture, I antennes Dimer nanoespace et plane « antenne-in-box ») et de démontrer son application dans l'amélioration de la détection d'une fluorescence seule molécule. Utilisation de l'analyse par spectroscopie de fluorescence d'explosion corrélation de fluorescence (FCS), les mesures TCSPC corrélées dans le temps et proches des simulations champ quantifier les volumes de détection de nanoantennas, les facteurs d'amélioration fluorescence et discuter des accélérations photodynamiques fluorescence médiée nanoantennas opticas. Des nanoantennas diélectriques à base de nanogap silico ont été proposées comme alternative dans l'amélioration de la détection par fluorescence de la diffusion de molécules uniques dans des solutions concentrées. En outre, l'utilisation de "plan d'antenne-in-box" dispositifs de résonance, nous étudions la dynamique de diffusion de phosphoéthanolamine et sphingomyéline dans la membrane plasmique des cellules vivantes et de discuter des résultats dans le contexte des radeaux lipidiques. Conjointement avec des expériences de réduction du cholestérol, nous fournissons des tests de division induits par le cholestérol dans le nanodomaine dans des diamètres plus petits de 10 nm et avec des temps caractéristiques de ~ 100 microsecondes