Quantitative fluorescence emission anisotropy microscopy for implementing homo-fluorescence resonance energy transfer measurements in living cells.

Quantitative fluorescence emission anisotropy microscopy reveals the organization of fluorescently labeled cellular components and allows their characterization in terms of changes in either rotational diffusion or homo-Förster's energy transfer characteristics in living cells. These properties provide insights into molecular organization, such as orientation, confinement, and oligomerization in situ. Here we elucidate how quantitative measurements of anisotropy using multiple microscope systems may be made by bringing out the main parameters that influence the quantification of fluorescence emission anisotropy. We focus on a variety of parameters that contribute to errors associated with the measurement of emission anisotropy in a microscope. These include the requirement for adequate photon counts for the necessary discrimination of anisotropy values, the influence of extinction ratios of the illumination source, the detector system, the role of numerical aperture, and excitation wavelength. All these parameters also affect the ability to capture the dynamic range of emission anisotropy necessary for quantifying its reduction due to homo-FRET and other processes. Finally, we provide easily implementable tests to assess whether homo-FRET is a cause for the observed emission depolarization.

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 cells. However, studies so far have 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, broadband aluminum-based nanoantennas carved at the apex of near-field probes are exploited to resolve nanoscale-dynamic molecular interactions on living cell membranes. Through multicolor excitation, the authors simultaneously recorded fluorescence fluctuations of dual-color labeled transmembrane receptors known to form nanoclusters. 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 the authors 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, the molecular diffusion within nanoclusters is resolved and distinguished from nanocluster diffusion. Spatiotemporal characterization of transient interactions between molecules is crucial to understand how they communicate with each other to regulate cell function. This work demonstrates the potential of broadband photonic antennas to study multi-molecular events and interactions in living cell membranes with unprecedented spatiotemporal resolution.

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.

Quantitative Control of GPCR Organization and Signaling by Endocytosis in Epithelial Morphogenesis.

Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. We show that GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, we probe the surface of the plasma membrane, and we show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and ß-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, we find higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. We identify that dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.

Large-Scale Arrays of Bowtie Nanoaperture Antennas for Nanoscale Dynamics in Living Cell Membranes.

We present a novel blurring-free stencil lithography patterning technique for high-throughput fabrication of large-scale arrays of nanoaperture optical antennas. The approach relies on dry etching through nanostencils to achieve reproducible and uniform control of nanoantenna geometries at the nanoscale, over millimeter-sizes in a thin aluminum film. We demonstrate the fabrication of over 400 000 bowtie nanoaperture (BNA) antennas on biocompatible substrates, having gap sizes ranging from (80 ± 5) nm down to (20 ± 10) nm. To validate their applicability on live cell research, we used the antenna substrates as hotspots of localized illumination to excite fluorescently labeled lipids on living cell membranes. The high signal-to-background afforded by the BNA arrays allowed the recording of single fluorescent bursts corresponding to the passage of freely diffusing individual lipids through hotspot excitation regions as small as 20 nm. Statistical analysis of burst length and intensity together with simulations demonstrate that the measured signals arise from the ultraconfined excitation region of the antennas. Because these inexpensive antenna arrays are fully biocompatible and amenable to their integration in most fluorescence microscopes, we foresee a large number of applications including the investigation of the plasma membrane of living cells with nanoscale resolution at endogenous expression levels.

Current approaches to studying membrane organization.

The local structure and composition of the outer membrane of an animal cell are important factors in the control of many membrane processes and mechanisms. These include signaling, sorting, and exo- and endocytic processes that are occurring all the time in a living cell. Paradoxically, not only are the local structure and composition of the membrane matters of much debate and discussion, the mechanisms that govern its genesis remain highly controversial. Here, we discuss a swathe of new technological advances that may be applied to understand the local structure and composition of the membrane of a living cell from the molecular scale to the scale of the whole membrane.

Hybrid photonic antennas for subnanometer multicolor localization and nanoimaging of single molecules.

Photonic antennas amplify and confine optical fields at the nanoscale offering excellent perspectives for nanoimaging and nanospectroscopy. Increased resolution beyond the diffraction limit has been demonstrated using a variety of antenna designs, but multicolor nanoscale imaging is precluded by their resonance behavior. Here we report on the design of a novel hybrid antenna probe based on a monopole nanoantenna engineered on a bowtie nanoaperture. The device combines broadband enhanced emission, extreme field confinement down to few nanometers, and zero-background illumination. We demonstrate simultaneous dual-color single molecule nanoimaging with 20 nm resolution and angstrom localization precision, corresponding to 10(3)-fold improvement compared to diffraction-limited optics. When interacting with individual molecules in the near-field, our innovative design enables the emission of 10(4) photon-counts per molecule in a 20 nm excitation region, allowing direct discrimination of spectrally distinct molecules separated by 2.1 ± 0.4 nm. We foresee that background-free nanolight sources will open new horizons in optical nanoscopy and fluorescence spectroscopy by providing multicolor detection of standard fluorescent molecules fully compatible with live cell research.

Priming by chemokines restricts lateral mobility of the adhesion receptor LFA-1 and restores adhesion to ICAM-1 nano-aggregates on human mature dendritic cells.

LFA-1 is a leukocyte specific ß2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.

Nanophotonic approaches for nanoscale imaging and single-molecule detection at ultrahigh concentrations.

Over the last decade, we have witnessed an outburst of many different optical techniques aimed at breaking the diffraction limit of light, providing super-resolution imaging on intact fixed cells. In parallel, single-molecule detection by means of fluorescence has become a common tool to investigate biological interactions at the molecular level both in vitro and in living cells. Despite these advances, visualization of dynamic events at relevant physiological concentrations at the nanometer scale remains challenging. In this review, we focus on recent advancements in the field of nanophotonics toward nanoimaging and single-molecule detection at ultrahigh sample concentrations. These approaches rely on the use of metal nanostructures known as optical antennas to localize and manipulate optical fields at the nanometer scale. We highlight examples on how different optical antenna geometries are being implemented for nanoscale imaging of cell membrane components. We also discuss different implementations of self-standing and two-dimensional antenna arrays for studying nanoscale dynamics in living cell membranes as well as detection of individual biomolecular interactions in the µM range for sensing applications.

PSF decomposition of nanoscopy images via Bayesian analysis unravels distinct molecular organization of the cell membrane.

The spatial organization of membrane receptors at the nanoscale has major implications in cellular function and signaling. The advent of super-resolution techniques has greatly contributed to our understanding of the cellular membrane. Yet, despite the increased resolution, unbiased quantification of highly dense features, such as molecular aggregates, remains challenging. Here we describe an algorithm based on Bayesian inference of the marker intensity distribution that improves the determination of molecular positions inside dense nanometer-scale molecular aggregates. We tested the performance of the method on synthetic images representing a broad range of experimental conditions, demonstrating its wide applicability. We further applied this approach to STED images of GPI-anchored and model transmembrane proteins expressed in mammalian cells. The analysis revealed subtle differences in the organization of these receptors, emphasizing the role of cortical actin in the compartmentalization of the cell membrane.

Biochemical and imaging methods to study receptor membrane organization and association with lipid rafts.

Lipid rafts, cell membrane domains with unique composition and properties, modulate the membrane distribution of receptors and signaling molecules facilitating the assembly of active signaling platforms. However, the underlying mechanisms that link signal transduction and lipid rafts are not fully understood, mainly because of the transient nature of these membrane assemblies. Several methods have been used to study the association of membrane receptors with lipid rafts. In the first part of this chapter, a description of how biochemical methods such as raft disruption by cholesterol depletion agents are useful in qualitatively establishing protein association with lipid rafts is presented. The second part of this chapter is dedicated to imaging techniques used to study membrane receptor organization and lipid rafts. We cover conventional approaches such as confocal microscopy to advanced imaging techniques such as homo-FRET microscopy and superresolution methods. For each technique described, their advantages and drawbacks are discussed.

A plasmonic 'antenna-in-box' platform for enhanced single-molecule analysis at micromolar concentrations.

Single-molecule fluorescence techniques are key for a number of applications, including DNA sequencing, molecular and cell biology and early diagnosis. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes. Although these nanoantennas enhance fluorescence brightness, large background signals and/or unspecific binding to the metallic surface have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel 'antenna-in-box' platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules.

Ultrabright bowtie nanoaperture antenna probes studied by single molecule fluorescence.

We report on a novel design for the fabrication of ultrabright bowtie nanoaperture antenna (BNA) probes to breach the intrinsic trade-off between power transmission and field confinement of circular nanoapertures as in near-field scanning optical microscopy (NSOM) or planar zero mode waveguides. The approach relies on the nanofabrication of BNAs at the apex of tapered optical fibers tuned to diameters close to their cutoff region, resulting in 10(3)× total improvement in throughput over conventional NSOM probes of similar confinement area. By using individual fluorescence molecules as optical nanosensors, we show for the first time nanoimaging of single molecules using BNA probes with an optical confinement of 80 nm, measured the 3D near-field emanating from these nanostructures and determined a ~6-fold enhancement on the single molecule signal. The broadband field enhancement, nanoscale confinement, and background free illumination provided by these nanostructures offer excellent perspectives as ultrabright optical nanosources for a full range of applications, including cellular nanoimaging, spectroscopy, and biosensing.

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion.

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes.

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.

Direct mapping of nanoscale compositional connectivity on intact cell membranes.

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains < 120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (< 150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy.

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

Imaging individual proteins and nanodomains on intact cell membranes with a probe-based optical antenna.

Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to <30 nm near the antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 +/- 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 +/- 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range.

A nanometer scale optical view on the compartmentalization of cell membranes.

For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of conventional light microscopy. Major developments, especially in the past few years, have demonstrated that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic measurements, a wealth of new information is now emerging regarding the compartmentalization of cell membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies, complementing the palette of biophysical techniques available nowadays.