Non-invasive assessment of axon radii via MRI bears great potential for clinical and neuroscience research as it is a main determinant of the neuronal conduction velocity. However, there is a lack of representative histological reference data at the scale of the cross-section of MRI voxels for validating the MRI-visible, effective radius (r(eff)). Because the current gold standard stems from neuroanatomical studies designed to estimate the bulk-determined arithmetic mean radius (r(arith)) on small ensembles of axons, it is unsuited to estimate the tail-weighted reff. We propose CNN-based segmentation on high-resolution, large-scale light microscopy (lsLM) data to generate a representative reference for r(eff). In a human corpus callosum, we assessed estimation accuracy and bias of r(arith) and r(eff). Furthermore, we investigated whether mapping anatomy-related variation of r(arith) and r(eff) is confounded by low-frequency variation of the image intensity, e.g., due to staining heterogeneity. Finally, we analyzed the error due to outstandingly large axons in r(eff). Compared to rarith, r(eff) was estimated with higher accuracy (maximum normalized-root-mean-square-error of r(eff): 8.5 %; r(arith): 19.5 %) and lower bias (maximum absolute normalized-mean-bias-error of r(eff): 4.8 %; r(arith): 13.4 %). While r(arith) was confounded by variation of the image intensity, variation of r(eff) seemed anatomy-related. The largest axons contributed between 0.8 % and 2.9 % to r(eff). In conclusion, the proposed method is a step towards representatively estimating r(eff) at MRI voxel resolution. Further investigations are required to assess generalization to other brains and brain areas with different axon radii distributions.

Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision.

Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein-protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Dynamic clustering of proteins on the nanoscale is a vital step in many signaling processes and other cellular functions. Localization microscopy techniques such as PALM and dSTORM provide methods to localize complexes to nanoscale resolution. Quantification of the number of underlying protein subunits is more difficult, however, due to the complex photophysics of the fluorophore labels. Re-activation, stochastic blinking, and incomplete detection all contribute to over- and under-counting artifacts. In order to generate an accurate quantification of protein subunit numbers we have developed a method based on fluorophore blinking kinetics captured in PALM microscopy. This approach takes advantage of both spatial and temporal information to form adaptive discrimination criteria and avoids both over- and under-counting quantification errors. The technique presented can correctly identify and quantify molecular clusters in simulated data with 98% accuracy. We will present additional data on the application of this technique in quantifying the nature of clusters of proteins involved in the immune synapse and immune signaling.

Focal adhesions are complex multi-protein structures that mediate cell adhesion and cell migration in multicellular organisms. Most of the protein components involved in focal adhesion formation have been identified, but a major challenge remains: determination of the spatial and temporal dynamics of adhesion proteins in order to understand the molecular mechanisms of adhesion assembly, maturation, signal regulation, and disassembly. Progress in this field has been hampered by the limited resolution of fluorescence microscopy. Recent advances have led to the development of super-resolution techniques including single-molecule localization microscopy (SMLM). Here, we discuss how the application of these techniques has revealed important new insights into focal adhesion structure and dynamics, including the first description of the three-dimensional nano-architecture of focal adhesions and of the dynamic exchange of integrins in focal adhesions. Hence, SMLM has contributed to the refinement of existing models of adhesions as well as the establishment of novel models, thereby opening new research directions. With current improvements in SMLM instrumentation and analysis, it has become possible to study cellular adhesions at the single-molecule level.

Significance Cell-expressed integrins mediate adhesion with other cells and with extracellular matrix and are essential for embryonic development and for controlling leukocyte migration in later life. Integrin adhesion depends on conformational change leading to activation, although it remains unknown exactly how integrins alter their conformational state and adhesion in response to guidance cues. We show that the guidance molecule plexinD1 controls clustering of integrins in patches on the cell membrane and that the activation state of individual integrins in these patches can be switched off by binding of sema3E to plexinD1. Disruption of this pathway causes abnormal thymocyte adhesion regulation and migration during development, leading to autoimmune phenomena.

Proteins will often dynamically transition between complexes of varying number of subunits depending on the interand intracellular environment. Labeling these subunits with fluorophores allows them to be localized to high precision with several super-resolution microscopy techniques, but quantifying the number of subunits in a sub-diffraction-limited area is complicated by the photophysical properties of the fluorophores. Blinking, re-activation, and complex kinetics all make identifying single molecules difficult.

The recent development of super-resolution techniques and particularly of single molecule localization microscopy (SMLM) has brought new insights in biology and it is now possible to study biological processes at the single molecule level. Coordinate-based techniques like PALM and dSTORM allow the precise localization of individual molecules in densely packed protein structures and can be used to characterize the spatial organization of proteins. Most cellular functions involve multiple components; therefore multi-color SMLM is an essential tool to address how interactions between different proteins determine biological function. In contrast with conventional fluorescence microscopy where image-based analysis can be applied to quantify protein colocalization, coordinate-based microscopy needs new strategies to quantify interactions between molecules imaged in different channels. In recent years, methodologies have been proposed to quantify dual colours SMLM data. However, none of them satisfyingly incorporates an evaluation of the spatial distribution to the quantification of the correlation between two channels. Molecular mechanisms such as T cell signalling [1] or in focal adhesions formation when a large number of proteins are recruited to a specific site would benefit from having both type of information available Here, we present a new analysis method to simultaneously quantify the degree of clustering and co-clustering in two-color PALM and dSTORM experiments. This method takes advantage of a cluster map analysis based on Ripley’s K function [2] combined with nearest neighbour analysis. We demonstrate the viability of this approach on different biological systems (T cell signalling and cell adhesion) and using various probes (Alexa 647, PS-CFP, PA-mCherry) to measure the molecular density of signalling clusters as well as their degree of co-clustering. Finally, we show that differences in the density and shape of clusters can affect the quantification of co-clustering.

Fluorescence lifetime correlation spectroscopy (FLCS) has been used to probe the influence of PEG-8000 on the fluidity of fluorescently labeled planar supported lipid bilayers on ozone plasma treated glass. The lipid membrane compositions examined were; DOPC, DOPC/DOPS (80/20 mol/mol) and DMPC, with and without cholesterol. The lateral diffusion coefficients (D) for supported lipid bilayer films of these layers without cholesterol were 7.9 +/- 0.2, 7.9 +/- 0.4 and 5.5 +/- 0.1 mu m(2) s(-1) respectively. The high fluidity reflected the super-hydrophilicity (contact angle of 0) of the ozone treated plasma glass substrate. Using DOPE conjugated Atto 655 as a probe, exposure of the lipid bilayer to a 30% wt/wt aqueous solution of PEG, followed by washing, dramatically increased the diffusion coefficients of the probe within the film. For example, the diffusion coefficient for the DOPC bilayer increases by nearly an order of magnitude to 51.4 +/- 2.6 mu m(2) s(-1). The autocorrelation curves for DOPC/DOPS (80/20 mol/mol) and DMPC bilayers required a two-component model for adequate fit of their behaviour yielding both fast and slow components of the diffusion. In all cases, when hydrophilic DOPE-Atto 655 was used as the probe, treatment of the lipid bilayer with PEG resulted in non-Brownian diffusion.Importantly, the observed diffusion behavior observed depends on the identity of probe. In contrast, when a hydrophobic probe (DOPE-NapthBodipy) was employed PEG showed relatively little impact on the observed diffusion rates. This was attributed to orientation of the reporter probe in the lipid bilayer and its aqueous interface. Specifically, DOPE-Atto 655 is believed to associate strongly with PEG mesh at the aqueous interface of the lipid bilayer, its diffusion strongly influenced by the structure in this region, whereas DOPE-NapthBodipy remains in the interior of the bilayer where it is relatively uninfluenced by PEG.

We focus on the functional role of small silver clusters in model hybrid systems involving peptides in the context of a new generation of nanostructured materials for biosensing. The optical properties of hybrids in the gas phase and at support will be addressed with the aim to bridge fundamental and application aspects. We show that extension and enhancement of absorption of peptides can be achieved by small silver clusters due to the interaction of intense intracluster excitations with the π-π* excitations of chromophoric aminoacids. Moreover, we demonstrate that the binding of a peptide to a supported silver cluster can be detected by the optical fingerprint. This illustrates that supported silver clusters can serve as building blocks for biosensing materials. Moreover, the clusters can be used simultaneously to immobilize biomolecules and to increase the sensitivity of detection, thus replacing the standard use of organic dyes and providing label-free detection. Complementary to that, we show that protected silver clusters containing a cluster core and a shell liganded by thiolates exhibit absorption properties with intense transitions in the visible regime which are also suitable for biosensing applications.

This article describes the synthesis and characterisation of fluorescent composite nanoparticles consisting of a silica core and a dextran shell. The silica core contains a rhodamine-based reference dye, which allows ratiometric measurements and the dextran shell is labelled with the Ca2+-sensitive dye Fluo-4. The nanoparticles have an average hydrodynamic diameter of 95 nm, good colloidal stability and show a 2.9-fold increase in fluorescence intensity upon binding to Ca2+ ions. The apparent dissociation constant of K'(d) approximate to 520 nM is well suited for measurements in the physiological range.

Silica shells are grown around colloidally synthesized gold nanorods (AuNRs) to form core–shell particles (AuNR@SiO 2 ) of variable occupancy, defined as the number of AuNRs per silica particle. Multiple AuNR occupancy within the silica shell, confirmed with high-resolution electron microscopy, is reflected in a redshift of the longitudinal plasmon mode of the nanorods due to multipolar coupling between AuNRs of a favored end–end orientation. In addition to the plasmon resonance that dominates their absorbance spectra, FL-AuNR@SiO 2 , core–shell particles incorporating a lipid probe (rhodamine-DOPE), can be monitored by their fluorescence and Raman signals. Optical and scanning electron microscopy (SEM) images are compared directly, enabling the correlation of spectroscopic characteristics with particle morphology. Raman and SEM images show that the most intense Raman signals come from aggregates of AuNRs trapped within the silica matrix. Biexponential fits to fluorescence decays indicate that competing mechanisms of quenching and fluorescence enhancement contribute to a reduced fluorescence lifetime of rhodamine-DOPE located near the AuNRs.

The formation of isolated [ (Trp-2H)+Ag9] + and [ (Tyr-2H)+Ag9] + amino-acid-Ag9 hybrids is reported. The photofragmentation yields of the aromatic amino acid-silver cluster hybrids, as well as those of the protonated tryptophan and tyrosine molecules ([Trp+H]+ and [Tyr+H]+) have been recorded. The fragmentation yields of the complexes are higher than the yields for [Trp+H]+and [Tyr+H]+ and present an extension of the fragmentation on the red side of the spectrum. The photofragmentation spectrum of [ Trp+Ag9] + was recently reported [Mitric et al., J. Chem. Phys. 127, 134301 (2007)]. While the optical spectra of substituted [ (Trp-2H)+Ag9] + and non-substituted [ Trp+Ag9] + complexes are very similar, a strong modification of the fragmentation channels between the two complexes is observed. The fragmentation channels are sensitive to the type of bonding in aromatic amino acid-silver cluster hybrids and can be used as fingerprints of structures.

Femtosecond pump-probe experiments are performed on flavin biomolecules isolated in an ion trap. Mass spectra of the photoinduced fragments show that the fragmentation pathways can be modified using two-color two-photon excitation. In particular, when an infrared probe pulse (810 nm) is added subsequent to the first excitation step (excitation of the S-1 state of flavin mononucleotide at 405 nm), branching ratios between lumichrome and lumiflavin production are inverted relative to the single excitation case.