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Understanding the pressure dependence of the nonlinear behavior of ultrasonically excited phospholipid-stabilized nanobubbles (NBs) is important for optimizing ultrasound exposure parameters for implementations of contrast enhanced ultrasound, critical to molecular imaging. The viscoelastic properties of the shell can be controlled by the introduction of membrane additives, such as propylene glycol as a membrane softener or glycerol as a membrane stiffener. We report on the production of high-yield NBs with narrow dispersity and different shell properties. Through precise control over size and shell structure, we show how these shell components interact with the phospholipid membrane, change their structure, affect their viscoelastic properties, and consequently change their acoustic response. A two-photon microscopy technique through a polarity-sensitive fluorescent dye, C-laurdan, was utilized to gain insights on the effect of membrane additives to the membrane structure. We report how the shell stiffness of NBs affects the pressure threshold (Pt) for the sudden amplification in the scattered acoustic signal from NBs. For narrow size NBs with 200 nm mean size, we find Pt to be between 123 and 245 kPa for the NBs with the most flexible membrane as assessed using C-Laurdan, 465-588 kPa for the NBs with intermediate stiffness, and 588-710 kPa for the NBs with stiff membranes. Numerical simulations of the NB dynamics are in good agreement with the experimental observations, confirming the dependence of acoustic response to shell properties, thereby substantiating further the development in engineering the shell of ultrasound contrast agents. The viscoelastic-dependent threshold behavior can be utilized for significantly and selectively enhancing the diagnostic and therapeutic ultrasound applications of potent narrow size NBs.

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane‐embedded polarity‐sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity‐sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity‐sensitive dyes C‐Laurdan or Di‐4‐ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.

Las membranas celulares son estructuras dinámicas complejas decomposición bioquímica muy diversa. Las interacciones específicas lípidolípidoy lípido-proteína pueden generar dominios estables o transientes decomposición y función biológica diferenciales. En este trabajo, abordamos el estudio de la regulación de la actividadde una proteína integral de membrana, la bomba de calcio de membranaplasmática (PMCA) por anfifilos que integran el sistema en el cual se lareconstituye. En micelas de lípidos y detergente, pudimos verificar que laactividad depende de la proporción relativa de estas especies y postulamosun modelo que explica esta dependencia en base al intercambio de anfifilosa nivel del dominio transmembrana. El análisis realizado con diversosmétodos espectroscópicos mostró que los cambios estructurales asociados ala activación por lípidos serían sutiles pero suficientes para transducir unaseñal de activación al dominio citoplasmático de PMCA. Utilizandoespectroscopía de correlación de fluorescencia y la sonda fluorescente Laurdan observamos que la actividad depende además de la fluidez delentorno y del tamaño de las micelas. Para extender este estudio al entornonatural de las proteínas -la membrana celular- evaluamos diversas sondasfluorescentes y pudimos determinar que la sonda C-Laurdan permiteestudiar por microscopía confocal la organización de membranasbiológicas. Cellular membranes are dynamic complex structures with a highlydiverse biochemical composition. Specific lipid-lipid and lipid-proteininteractions can generate transient or stable domains of particularcomposition and biological function. In this work, we study the regulation of the activity of an integralmembrane protein, the plasma membrane calcium pump (PMCA), byphospholipids that make up the reconstitution system. Working in a mixed micelles lipid/detergent system, we verify thatthe activity depends on the relative proportion of these species and weposited a model that explains this relationship by the exchange betweenamphiphiles at the protein transmembrane domain. The analysis performedby spectroscopic methods showed that the structural changes associatedwith the activation by lipids are subtle but enough to transduce anactivation signal to the cytoplasmic domain of PMCA. Using fluorescencecorrelation spectroscopy and the fluorescent probe Laurdan, we observedthat the activity is also related to micelle size and the fluidity of the proteinlipidic microenvironment. To extend this study to the natural environmentof the protein –the cellular membrane- we assayed the performance ofseveral fluorescent probes and we could determine that the probe CLaurdanallows studying the organization of biological membranes byconfocal microscopy. Fil: Dodes Traian, Martín Miguel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Many biologically important processes and phcnomena in lipid membranes are still not fully understood. The presence of ions and water molœules has a significant influence on the structural and dynamical properties of lipid bilayers. Fluorescent techniques are versatile tools for studying the lipid membranes, because the fluorescence emission is strongly sensitive to dye environment. We have conducted fluorescent solvent relaxation (SR) experiments to explore the hydration and mobility properties in lipid membranes in the presence of different chaotropic ions. We have also carried out Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations for supporting the SR experiments. SR experiments show that small cation (Na+) is attracted to the membrane and increases rigidity ofbilayer, while larger cations (NH/, Cs+) should not. Large anions (CI04·, SCN') adsorl, at the membrane interface more easily than smaller ones (Cl') and significantly change tl!e mobility and hydration of the headgroup region oflipid bilayer. SR study ofhydrophobic part of the membrane show that SR processes are complex there and reflect botl!: faster, intramolecular (torsional relaxation or fonnation of charge transfer state) and slower, intermolecular (SR) relaxation processes. QM calculatiom were used to create force-field for three fluorescent dyes (Prodan, Laurdan and C-laurdan). MD simulations allow detennining position of the dye in the lipid membrane in the ground state and after excitation and reproduce correctly SR timescale- ps in water and ns in the membrane. MD simulations extend the capabilities of SR method and allow observing the behaviour of individual molecules.; De nombreux processus biologiques liés aux membranes cellulaires lipidiques sont encore très mal connus. La présence d'eau et d'ions à l'interface influence les propriétés structurelles et dynamiques de la bicouche lipidique. Les techniques de fluorescence sont très utiles pour étudier les membranes en raison de la grande sensibilité des sondes à leur environnement. Nous avons utilisé la technique de relaxation de solvant (SR) pour explorer l'hydratation et la mobilité de l'eau. Nous avons également réalisé des calculs quantiques (QM) et des dynamiques moléculaires (DM) pour étayer nos expériences. Les résultats SR montrent qu'un petit cation (Na+) est très attiré par la membrane et augmente sa rigidité à l'opposé des cations (NH4+, Cs+) plus gros. Les anions (CI04-, SCN-) s'adsorbent à l'interface plus facilement que Cl-. Ces anions changent la mobilité et l'hydratation des têtes polaires des lipides de la bicouche. Les études SR de la zone hydrophobe de la membrane montrent que les processus de relaxation sont ici très complexes. lis reflètent des processus rapides intramoléculaire (relaxation de torsion, transferts de charge) et des processus intermoléculaires lents. Les calculs QM ont permis de créer les champs de force de trois sondes fluorescentes (Prodan, Laurdan et C-laurdan). Les simulations DM ont permis de déterminer les positions des sondes dans une membrane DOPC. La modélisation reproduit correctement les résultats SR, en particulier les temps de relaxation : de l'ordre de la ps en solvant aqueux et de la ns dans la membrane. Les simulations MD sont complémentaires des méthodes SR et permettent de surveiller le comportement de molécules uniques.