A local view on single and coupled molecules

The paper focuses on a novel approach to reveal ultrafast dynamics in single molecules. The main strength of the approach is towards ultrafast processes in extended multi-chromophoric molecular assemblies. Excitonically coupled systems consisting of 2 and 3 rigidly linked perylene-diimide units in a head to tail configuration are studied. Superradiance and inhibited intramolecular decay are observed and discrete jumps in femtosecond response upon break-up of the strong coupling are revealed

Weak ergodicity breaking of receptor motion in living cells stemming from random diffusivity

Molecular transport in living systems regulates numerous processes underlying biological function. Although many cellular components exhibit anomalous diffusion, only recently has the subdiffusive motion been associated with nonergodic behavior. These findings have stimulated new questions for their implications in statistical mechanics and cell biology. Is nonergodicity a common strategy shared by living systems? Which physical mechanisms generate it? What are its implications for biological function? Here, we use single particle tracking to demonstrate that the motion of DC-SIGN, a receptor with unique pathogen recognition capabilities, reveals nonergodic subdiffusion on living cell membranes. In contrast to previous studies, this behavior is incompatible with transient immobilization and therefore it can not be interpreted according to continuous time random walk theory. We show that the receptor undergoes changes of diffusivity, consistent with the current view of the cell membrane as a highly dynamic and diverse environment. Simulations based on a model of ordinary random walk in complex media quantitatively reproduce all our observations, pointing toward diffusion heterogeneity as the cause of DC-SIGN behavior. By studying different receptor mutants, we further correlate receptor motion to its molecular structure, thus establishing a strong link between nonergodicity and biological function. These results underscore the role of disorder in cell membranes and its connection with function regulation. Due to its generality, our approach offers a framework to interpret anomalous transport in other complex media where dynamic heterogeneity might play a major role, such as those found, e.g., in soft condensed matter, geology and ecology.Comment: 27 pages, 5 figure

Broadband plasmonic nanoantennas for multi-color nanoscale dynamics in living cells

Recently, the implementation of plasmonic nanoantennas has opened new possibilities to investigate the nanoscale dynamics of individual biomolecules in living cell. However, studies have yet been restricted to single molecular species as the narrow wavelength resonance of gold-based nanostructures precludes the simultaneous interrogation of different fluorescently labeled molecules. Here we exploited broadband aluminum-based nanoantennas carved at the apex of near-field probes to resolve nanoscale-dynamic molecular interactions on intact living cell membranes. Through multicolor excitation, we simultaneously recorded fluorescence fluctuations of dual-color labeled transmembrane receptors known to form nanoclusters in living cells. Fluorescence cross-correlation studies revealed transient interactions between individual receptors in regions of ~60 nm. Moreover, the high signal-to-background ratio provided by the antenna illumination allowed us to directly detect fluorescent bursts arising from the passage of individual receptors underneath the antenna. Remarkably, by reducing the illumination volume below the characteristic receptor nanocluster sizes, we resolved molecular diffusion within nanoclusters and distinguished it from nanocluster diffusion. Spatiotemporal characterization of transient interactions between molecules is crucial to understand how they communicate with each other to regulate cell function. Our work demonstrates the potential of broadband photonic antennas to study multi-molecular events and interactions in living cell membranes with unprecedented spatiotemporal resolution

Enhancing Magnetic Light Emission with All-Dielectric Optical Nanoantennas

Electric and magnetic optical fields carry the same amount of energy. Nevertheless, the efficiency with which matter interacts with electric optical fields is commonly accepted to be at least 4 orders of magnitude higher than with magnetic optical fields. Here, we experimentally demonstrate that properly designed photonic nanoantennas can selectively manipulate the magnetic versus electric emission of luminescent nanocrystals. In particular, we show selective enhancement of magnetic emission from trivalent europium-doped nanoparticles in the vicinity of a nanoantenna tailored to exhibit a magnetic resonance. Specifically, by controlling the spatial coupling between emitters and an individual nanoresonator located at the edge of a near field optical scanning tip, we record with nanoscale precision local distributions of both magnetic and electric radiative local densities of states (LDOS). The map of the radiative LDOS reveals the modification of both the magnetic and electric quantum environments induced by the presence of the nanoantenna. This manipulation and enhancement of magnetic light-matter interaction by means of nanoantennas opens up new possibilities for the research fields of opto-electronics, chiral optics, nonlinear&nano-optics, spintronics and metamaterials, amongst others.Peer ReviewedPostprint (author's final draft

A DNA Origami Platform for Quantifying Protein Copy Number in Super-Resolution

Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.The authors gratefully acknowledge financial support fromthe European Commission (FP7-ICT-2011-7, grant number 288263), Erasmus Mundus Doctorate Program Europhoton-ics (grant number 159224-1-2009-1-FR-ERA MUNDUS-EMJD), Spanish Ministry of Economy and Competi-tiveness (“Severo Ochoa” Programme for Centres of Excellence in Research & Development SEV-2015-0522,and FIS2014-56107-R grants), Generalitat de Catalunyathrough the CERCA program, Italian Ministry of Uni-versity and Research (FIRB “Futuro in Ricerca” 2013grant n. RBFR13V4M2 and Flagship Project NANOMAX),Fundaci ́o Privada CELLEX (Barcelona), Ente Cassa diRisparmio di Firenze, Human Frontier Science Program (GARGP0027/2012) and LaserLab Europe 4 (GA 654148). C.M.acknowledges funding from the Spanish Ministry of Econ-omy and Competitiveness (MINECO) and the EuropeanSocial Fund (ESF) through the Ram ́on y Cajal program 2015(RYC-2015-17896).Peer ReviewedPostprint (author's final draft

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

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nanoclusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular domain. This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular domain and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nanoclusters localized to a meso-scale meshwork pattern

Hybrid plasmonic nanostructures for enhanced single-molecule detection sensitivity

Biosensing applications based on fluorescence detection often require single-molecule sensitivity in the presence of strong background signals. Plasmonic nanoantennas are particularly suitable for these tasks, as they can confine and enhance light in volumes far below the diffraction limit. The recently introduced antenna-in-box (AiB) platforms achieved high single-molecule detection sensitivity at high fluorophore concentrations by placing gold nanoantennas in a gold aperture. However, hybrid AiB platforms with alternative aperture materials such as aluminum promise superior performance by providing better background screening. Here, we report on the fabrication and optical characterization of hybrid AiBs made of gold and aluminum for enhanced single-molecule detection sensitivity. We computationally optimize the optical properties of AiBs by controlling their geometry and materials and find that hybrid nanostructures not only improve signal-to-background ratios but also provide additional excitation intensity and fluorescence enhancements. We further establish a two-step electron beam lithography process to fabricate hybrid material AiB arrays with high reproducibility and experimentally validate the higher excitation and emission enhancements of the hybrid nanostructures as compared to their gold counterpart. We foresee that biosensors based on hybrid AiBs will provide improved sensitivity beyond the capabilities of current nanophotonic sensors for a plethora of biosensing applications ranging from multicolor fluorescence detection to label-free vibrational spectroscopy