Fluorescent diheteroarylethenes as probes for fluorescence microscopy

Las técnicas modernas de microscopía de fluorescencia requieren sondas cuyoscomportamientos fotoquímicos, combinados con las características ópticas del equipamiento,permitan resolver estructuras de dimensiones menores al límite impuesto por la difracciónnatural de la luz. Además de ser brillantes, estables, pequeñas y compatibles con el mediobiológico, las sondas ideales fluorescen y se tornan oscuras de manera controlada y conocida. En esta tesis se diseñaron nuevas sondas basadas en los diheteroariletenos oxidados (DHAOs)como plataforma de fluorescencia controlada. Los DHAOs son compuestos fotocrómicos que presentan dos isómeros fotoestables, unofluorescente y uno oscuro, alcanzados por irradiación con luz ultravioleta y visiblerespectivamente. Las propiedades espectroscópicas de los DHAOs se optimizaron estudiandoel efecto de sustituyentes dadores y aceptores de electrones sobre las mismas. La demandantecaracterización espectrofotométrica de los DHAOs se abordó con un espectrofotómetro yfotomodulador de construcción propia, el cual permitió automatizar las mediciones y explorarsistemáticamente diferentes condiciones de fotoconversión. Se descubrió que al aumentar elcarácter dador de electrones del sustituyente, se estabiliza particularmente el estado excitadodel isómero fluorescente, resultando en un aumento del desplazamiento de Stokes y unpronunciado solvatocromismo del fluoróforo. El solvatocromismo se utilizó para estudiar elcambio de polaridad del entorno de monómeros de α-sinucleína durante su agregación. A fin de conservar la fluoresencia fotomodulable de los DHAOs en agua, se explorarondiversas estrategias de solubilización con modificaciones covalentes y no-covalentes. Lainclusión en cavidades de ciclodextrina, la adsorción a albúmina, y la funcionalización congrupos hidrofílicos sirvieron para solubilizar efectivamente a los DHAOs en medios acuosos. Las propiedades de fluorescencia y fotoconversión se retuvieron exitosamente dentro de lacavidad de las proteínas de unión de ácidos grasos y mediante la conjugación a entidadesprotéicas de mayor tamaño, indicando la preferencia de los DHAOs a un entorno protegido delagua. Anticuerpos secundarios conjugados a DHAOs se utilizaron para marcar β-tubulinaen neuronas, y se lograron fotoconvertir selectivamente distintas áreas de las muestras enun microscopio que incorpora un arreglo programable para generar patrones espaciales deexcitación. El particular comportamiento fotoquímico de los diheteroariletenos permitió desarrollar unaestrategia para aumentar la resolución microscópica combinando DHAOs con fotocrómicosno-fluorescentes. Utilizando el fenómeno de transferencia de energía por resonancia de Förster, se postulan sondas que requieren de múltiples estímulos para la activación de laemisión, disminuyendo el volúmen de activación por debajo del límite de difracción de laluz y aumentando la resolución axial. La aplicación de las sondas sintetizadas y la propuestade nuevas estrategias relacionadas con los DHAOs denotan el potencial que tienen los mismosen la búsqueda de mayor resolución en la microscopía de fluorescencia.Modern fluorescence microscopy techniques require probes whose photochemical behaviour,combined with the optical characteristics of the microscope, enable the resolution of structuressmaller than the natural diffraction limit of light. In addition to being bright, stable, small andcompatible with the biological environment, the ideal probe’s fluorescence is activated anddeactivated in a controlled and predictable manner. New probes were designed using oxidizeddiheteroarylethenes (ODHAs) as a platform for controlled fluorescence and are presented inthis thesis. ODHAs are photochromic compounds with two photostable isomers, one fluorescent andone dark, which are formed upon irradiation with ultraviolet and visible light, respectively. In order to optimize their spectroscopic and photochemical properties, the ODHAs weremodified with electron-donating and -withdrawing substituents. The demanding spectroscopiccharacterization of the photochromes was tackled with a custom built spectrophotometerand photoconverter, which enabled automatization of the measurements and the systematicexploration of varying photoconversion conditions. By increasing the electron-donatingcharacter of the substituents, the excited state of the fluorescent isomer is particularlystabilized, resulting in an increase in the Stokes shift and pronounced solvatochromism ofthe fluorophore. The solvatochromic ODHA was used to probe the changes in local polarityaround monomers of the amyloid protein α-synuclein during their aggregation. Diverse solubilization strategies involving both covalent and non-covalent modificationswere employed with the aim of conserving the photoswitchable fluorescence of ODHAs inwater. Inclusion in cyclodextrin cavities, adsorption onto albumin, and functionalization withhydrophilic groups all served to effectively solubilize the ODHAs in aqueous solutions. Thefluorescence and photoconversion properties were conserved inside the cavities of fatty acidbinding proteins and by conjugation to larger protein entities, indicating the preference ofthe ODHAs for surroundings devoid of water. Secondary antibodies conjugated to ODHAswere used to immunostain β-tubulin in fixed neurons, and different areas of the samplewere selectively photoconverted using structured illumination with a structured illumination (programmable array) microscope. A new strategy for increasing microscopic resolution was developed, harnessing the uniquephotochemical behaviour of diheteroarylethenes by combining ODHAs with non-fluorescentphotochromes. The proposed probes require multiple stimuli for activation of the Försterresonance energy transfer-modulated emission, reducing the activation volume below thediffraction limit and increasing axial resolution. The application of the synthesized probesand the description of novel strategies related to ODHAs denote their potential in the searchfor increased resolution in fluorescence microscopy.Fil: Gillanders, Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Photoswitchable fluorescent diheteroarylethenes: substituent effects on photochromic and solvatochromic properties

Photoswitchable fluorescent diheteroarylethenes are promising candidates for applications in super-resolution molecular localization fluorescence microscopy thanks to their high quantum yields and fatigue-resistant photoswitching characteristics. We have studied the effect of varying substituents on the photophysical properties of six sulfone derivatives of diheteroarylethenes, which display fluorescence in one (closed form) of two thermally stable photochromic states. Electron-donating substituents displace the absorption and emission spectra towards the red without substantially affecting the fluorescence quantum yields. Furthermore, ethoxybromo, a very electron-donating substituent, stabilizes the excited state of the closed isomer to the extent of almost entirely inhibiting its cycloreversion. Multi-parameter Hammett correlations indicate a relationship between the emission maxima and electron-donating character, providing a useful tool in the design of future photochromic molecules. Most of the synthesized compounds exhibit small bathochromic shifts and shorter fluorescence lifetimes with an increase in solvent polarity. However, the ethoxybromo-substituted fluorescent photochrome is unique in its strong solvatochromic behaviour, constituting a photoactivatable (photochromic), fluorescent and highly solvatochromic small organic compound. The Catalán formalism identified solvent dipolarity as the principal basis of the solvatochromism, reflecting the highly polarized nature of this molecule.Fil: Gillanders, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Giordano, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; AlemaniaFil: Diaz, Sebastian Andres. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; AlemaniaFil: Jovin, Tomás. Max Planck Institute for Biophysical Chemistry. Laboratory of Cellular Dynamics; AlemaniaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentin

Fluorescent diheteroarylethenes as probes for fluorescence microscopy

Las técnicas modernas de microscopía de fluorescencia requieren sondas cuyoscomportamientos fotoquímicos, combinados con las características ópticas del equipamiento,permitan resolver estructuras de dimensiones menores al límite impuesto por la difracciónnatural de la luz. Además de ser brillantes, estables, pequeñas y compatibles con el mediobiológico, las sondas ideales fluorescen y se tornan oscuras de manera controlada y conocida. En esta tesis se diseñaron nuevas sondas basadas en los diheteroariletenos oxidados (DHAOs)como plataforma de fluorescencia controlada. Los DHAOs son compuestos fotocrómicos que presentan dos isómeros fotoestables, unofluorescente y uno oscuro, alcanzados por irradiación con luz ultravioleta y visiblerespectivamente. Las propiedades espectroscópicas de los DHAOs se optimizaron estudiandoel efecto de sustituyentes dadores y aceptores de electrones sobre las mismas. La demandantecaracterización espectrofotométrica de los DHAOs se abordó con un espectrofotómetro yfotomodulador de construcción propia, el cual permitió automatizar las mediciones y explorarsistemáticamente diferentes condiciones de fotoconversión. Se descubrió que al aumentar elcarácter dador de electrones del sustituyente, se estabiliza particularmente el estado excitadodel isómero fluorescente, resultando en un aumento del desplazamiento de Stokes y unpronunciado solvatocromismo del fluoróforo. El solvatocromismo se utilizó para estudiar elcambio de polaridad del entorno de monómeros de α-sinucleína durante su agregación. A fin de conservar la fluoresencia fotomodulable de los DHAOs en agua, se explorarondiversas estrategias de solubilización con modificaciones covalentes y no-covalentes. Lainclusión en cavidades de ciclodextrina, la adsorción a albúmina, y la funcionalización congrupos hidrofílicos sirvieron para solubilizar efectivamente a los DHAOs en medios acuosos. Las propiedades de fluorescencia y fotoconversión se retuvieron exitosamente dentro de lacavidad de las proteínas de unión de ácidos grasos y mediante la conjugación a entidadesprotéicas de mayor tamaño, indicando la preferencia de los DHAOs a un entorno protegido delagua. Anticuerpos secundarios conjugados a DHAOs se utilizaron para marcar β-tubulinaen neuronas, y se lograron fotoconvertir selectivamente distintas áreas de las muestras enun microscopio que incorpora un arreglo programable para generar patrones espaciales deexcitación. El particular comportamiento fotoquímico de los diheteroariletenos permitió desarrollar unaestrategia para aumentar la resolución microscópica combinando DHAOs con fotocrómicosno-fluorescentes. Utilizando el fenómeno de transferencia de energía por resonancia de Förster, se postulan sondas que requieren de múltiples estímulos para la activación de laemisión, disminuyendo el volúmen de activación por debajo del límite de difracción de laluz y aumentando la resolución axial. La aplicación de las sondas sintetizadas y la propuestade nuevas estrategias relacionadas con los DHAOs denotan el potencial que tienen los mismosen la búsqueda de mayor resolución en la microscopía de fluorescencia.Modern fluorescence microscopy techniques require probes whose photochemical behaviour,combined with the optical characteristics of the microscope, enable the resolution of structuressmaller than the natural diffraction limit of light. In addition to being bright, stable, small andcompatible with the biological environment, the ideal probe’s fluorescence is activated anddeactivated in a controlled and predictable manner. New probes were designed using oxidizeddiheteroarylethenes (ODHAs) as a platform for controlled fluorescence and are presented inthis thesis. ODHAs are photochromic compounds with two photostable isomers, one fluorescent andone dark, which are formed upon irradiation with ultraviolet and visible light, respectively. In order to optimize their spectroscopic and photochemical properties, the ODHAs weremodified with electron-donating and -withdrawing substituents. The demanding spectroscopiccharacterization of the photochromes was tackled with a custom built spectrophotometerand photoconverter, which enabled automatization of the measurements and the systematicexploration of varying photoconversion conditions. By increasing the electron-donatingcharacter of the substituents, the excited state of the fluorescent isomer is particularlystabilized, resulting in an increase in the Stokes shift and pronounced solvatochromism ofthe fluorophore. The solvatochromic ODHA was used to probe the changes in local polarityaround monomers of the amyloid protein α-synuclein during their aggregation. Diverse solubilization strategies involving both covalent and non-covalent modificationswere employed with the aim of conserving the photoswitchable fluorescence of ODHAs inwater. Inclusion in cyclodextrin cavities, adsorption onto albumin, and functionalization withhydrophilic groups all served to effectively solubilize the ODHAs in aqueous solutions. Thefluorescence and photoconversion properties were conserved inside the cavities of fatty acidbinding proteins and by conjugation to larger protein entities, indicating the preference ofthe ODHAs for surroundings devoid of water. Secondary antibodies conjugated to ODHAswere used to immunostain β-tubulin in fixed neurons, and different areas of the samplewere selectively photoconverted using structured illumination with a structured illumination (programmable array) microscope. A new strategy for increasing microscopic resolution was developed, harnessing the uniquephotochemical behaviour of diheteroarylethenes by combining ODHAs with non-fluorescentphotochromes. The proposed probes require multiple stimuli for activation of the Försterresonance energy transfer-modulated emission, reducing the activation volume below thediffraction limit and increasing axial resolution. The application of the synthesized probesand the description of novel strategies related to ODHAs denote their potential in the searchfor increased resolution in fluorescence microscopy.Fil: Gillanders, Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

ESIPT and FRET probes for monitoring nanoparticle polymer coating stability

Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.Fil: Azcárate, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Diaz, Sebastian Andres. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Fauerbach, Jonathan Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Gillanders, Florencia. Max Planck Institute For Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias ; ArgentinaFil: Rubert, Aldo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Jares, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Jovin, Thomas M.. Max Planck Institute For Biophysical Chemistry; AlemaniaFil: Fonticelli, Mariano Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin