Single-emitter super-resolved imaging of radiative decay rate enhancement in dielectric gap nanoantennas.

High refractive index dielectric nanoantennas strongly modify the decay rate via the Purcell effect through the design of radiative channels. Due to their dielectric nature, the field is mainly confined inside the nanostructure and in the gap, which is hard to probe with scanning probe techniques. Here we use single-molecule fluorescence lifetime imaging microscopy (smFLIM) to map the decay rate enhancement in dielectric GaP nanoantenna dimers with a median localization precision of 14 nm. We measure, in the gap of the nanoantenna, decay rates that are almost 30 times larger than on a glass substrate. By comparing experimental results with numerical simulations we show that this large enhancement is essentially radiative, contrary to the case of plasmonic nanoantennas, and therefore has great potential for applications such as quantum optics and biosensing.

3D topographies promote macrophage M2d-Subset differentiation.

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

3D-Architected Alkaline-Earth Perovskites.

3D ceramic architectures are captivating geometrical features with an immense demand in optics. In this work, an additive manufacturing (AM) approach for printing alkaline-earth perovskite 3D microarchitectures is developed. The approach enables custom-made photoresists suited for two-photon lithography, permitting the production of alkaline-earth perovskite (BaZrO3 , CaZrO3 , and SrZrO3 ) 3D structures shaped in the form of octet-truss lattices, gyroids, or inspired architectures like sodalite zeolite, and C60 buckyballs with micrometric and nanometric feature sizes. Alkaline-earth perovskite morphological, structural, and chemical characteristics are studied. The optical properties of such perovskite architectures are investigated using cathodoluminescence and wide-field photoluminescence emission to estimate the lifetime rate and defects in BaZrO3 , CaZrO3 , and SrZrO3 . From a broad perspective, this AM methodology facilitates the production of 3D-structured mixed oxides. These findings are the first steps toward dimensionally refined high-refractive-index ceramics for micro-optics and other terrains like (photo/electro)catalysis.

Real-time detection of virus antibody interaction by label-free common-path interferometry.

Viruses have a profound influence on all forms of life, motivating the development of rapid and minimally invasive methods for virus detection. In this study, we present a novel methodology that enables quantitative measurement of the interaction between individual biotic nanoparticles and antibodies in solution. Our approach employs a label-free, full-field common-path interferometric technique to detect and track biotic nanoparticles and their interactions with antibodies. It is based on the interferometric detection of light scattered by viruses in aqueous samples for the detection of individual viruses. We employ single-particle tracking analysis to characterize the size and properties of the detected nanoparticles, and to monitor the changes in their diffusive mobility resulting from interactions. To validate the sensitivity of our detection approach, we distinguish between particles having identical diffusion coefficients but different scattering signals, using DNA-loaded and DNA-devoid capsids of the Escherichia coli T5 virus phage. In addition, we have been able to monitor, in real time, the interaction between the bacteriophage T5 and purified antibodies targeting its major capsid protein pb8, as well as between the phage SPP1 and nonpurified anti-SPP1 antibodies present in rabbit serum. Interestingly, these virus-antibody interactions are observed within minutes. Finally, by estimating the number of viral particles interacting with antibodies at different concentrations, we successfully quantify the dissociation constant Kd of the virus-antibody reaction using single-particle tracking analysis.

Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.

BACKGROUND: Centromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context. RESULTS: Using transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC. CONCLUSION: Our cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

Cramér-Rao analysis of lifetime estimations in time-resolved fluorescence microscopy.

Measuring the lifetime of fluorescent emitters by time-correlated single photon counting (TCSPC) is a routine procedure in many research areas spanning from nanophotonics to biology. The precision of such measurement depends on the number of detected photons but also on the various sources of noise arising from the measurement process. Using Fisher information theory, we calculate the lower bound on the precision of lifetime estimations for mono-exponential and bi-exponential distributions. We analyse the dependence of the lifetime estimation precision on experimentally relevant parameters, including the contribution of a non-uniform background noise and the instrument response function (IRF) of the setup. We also provide an open-source code to determine the lower bound on the estimation precision for any experimental conditions. Two practical examples illustrate how this tool can be used to reach optimal precision in time-resolved fluorescence microscopy.

Multi-scale tracking reveals scale-dependent chromatin dynamics after DNA damage.

The dynamic organization of genes inside the nucleus is an important determinant for their function. Using fast DNA tracking microscopy in S. cerevisiae cells and improved analysis of mean square displacements, we quantified DNA motion at time scales ranging from 10 milliseconds to minute and found that following DNA damage, DNA exhibits distinct sub-diffusive regimes. In response to double-strand breaks, chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales. This effect is even more pronounced at the site of damage. Such a pattern of dynamics is consistent with a global increase in chromatin persistence length in response to DNA damage. Scale-dependent nuclear exploration is regulated by the Rad51 repair protein, both at the break and throughout the genome. We propose a model in which stiffening of the damaged ends by the repair complex, combined with global increased stiffness, act like a "needle in a ball of yarn", enhancing the ability of the break to traverse the chromatin meshwork.

Single molecule study of non-specific binding kinetics of LacI in mammalian cells.

Many key cellular processes are controlled by the association of DNA-binding proteins (DBPs) to specific sites. The kinetics of the search process leading to the binding of DBPs to their target locus are largely determined by transient interactions with non-cognate DNA. Using single-molecule microscopy, we studied the dynamics and non-specific binding to DNA of the Lac repressor (LacI) in the environment of mammalian nuclei. We measured the distribution of the LacI-DNA binding times at non-cognate sites and determined the mean residence time to be τ(1D) = 182 ms. This non-specific interaction time, measured in the context of an exogenous system such as that of human U2OS cells, is remarkably different compared to that reported for the LacI in its native environment in E. coli (<5 ms). Such a striking difference (more than 30 fold) suggests that the genome, its organization, and the nuclear environment of mammalian cells play important roles on the dynamics of DBPs and their non-specific DNA interactions. Furthermore, we found that the distribution of off-target binding times follows a power law, similar to what was reported for TetR in U2OS cells. We argue that a possible molecular origin of such a power law distribution of residence times is the large variability of non-cognate sequences found in the mammalian nucleus by the diffusing DBPs.

Geometry of the nucleus: a perspective on gene expression regulation.

Gene expression control results from the combined interactions of the nearly hundred proteins forming the pre-initiation complex, thousands of transcription regulators, and genomic DNA. In the recent years, new technologies have revealed several key aspects of nuclear spatial organization that showed a fine interplay between the function of nuclear proteins, their 3D organization, and their dynamics. Here we review several concepts that link biochemical reactivity in the nucleus to its 3D spatial organization. We present the analogies between the emerging understanding of nuclear organization in the field of cell biology, and the more established disciplines of heterogeneous catalysis and the physics of random walks. We provide several recent examples showing how nuclear geometry affects protein reactivity in the nucleus.

Accessing the third dimension in localization-based super-resolution microscopy.

Only a few years after its inception, localization-based super-resolution microscopy has become widely employed in biological studies. Yet, it is primarily used in two-dimensional imaging and accessing the organization of cellular structures at the nanoscale in three dimensions (3D) still poses important challenges. Here, we review optical and computational techniques that enable the 3D localization of individual emitters and the reconstruction of 3D super-resolution images. These techniques are grouped into three main categories: PSF engineering, multiple plane imaging and interferometric approaches. We provide an overview of their technical implementation as well as commentary on their applicability. Finally, we discuss future trends in 3D localization-based super-resolution microscopy.

Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus.

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.

The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion.

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.

Single cell correlation fractal dimension of chromatin: a framework to interpret 3D single molecule super-resolution.

Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state.

Real-time dynamics of RNA polymerase II clustering in live human cells.

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites.

The strength of synaptic transmission is controlled by the number and activity of neurotransmitter receptors. However, little is known about absolute numbers and densities of receptor and scaffold proteins and the stoichiometry of molecular interactions at synapses. Here, we conducted three-dimensional and quantitative nanoscopic imaging based on single-molecule detections to characterize the ultrastructure of inhibitory synapses and to count scaffold proteins and receptor binding sites. We observed a close correspondence between the spatial organization of gephyrin scaffolds and glycine receptors at spinal cord synapses. Endogenous gephyrin was clustered at densities of 5,000-10,000 molecules/µm(2). The stoichiometry between gephyrin molecules and receptor binding sites was approximately 1:1, consistent with a two-dimensional scaffold in which all gephyrin molecules can contribute to receptor binding. The competition of glycine and GABAA receptor complexes for synaptic binding sites highlights the potential of single-molecule imaging to quantify synaptic plasticity on the nanoscopic scale.

PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking.

We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.

Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy.

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.

Lateral diffusion on tubular membranes: quantification of measurements bias.

Single Particle Tracking (SPT) is a powerful technique for the analysis of the lateral diffusion of the lipid and protein components of biological membranes. In neurons, SPT allows the study of the real-time dynamics of receptors for neurotransmitters that diffuse continuously in and out synapses. In the simplest case where the membrane is flat and is parallel to the focal plane of the microscope the analysis of diffusion from SPT data is relatively straightforward. However, in most biological samples the membranes are curved, which complicates analysis and may lead to erroneous conclusions as for the mode of lateral diffusion. Here we considered the case of lateral diffusion in tubular membranes, such as axons, dendrites or the neck of dendritic spines. Monte Carlo simulations allowed us to evaluate the error in diffusion coefficient (D) calculation if the curvature is not taken into account. The underestimation is determined by the diameter of the tubular surface, the frequency of image acquisition and the degree of mobility itself. We found that projected trajectories give estimates that are 25 to 50% lower than the real D in case of 2D-SPT over the tubular surface. The use of 3D-SPT improved the measurements if the frequency of image acquisition was fast enough in relation to the mobility of the molecules and the diameter of the tube. Nevertheless, the calculation of D from the components of displacements in the axis of the tubular structure gave accurate estimate of D, free of geometrical artefacts. We show the application of this approach to analyze the diffusion of a lipid on model tubular membranes and of a membrane-bound GFP on neurites from cultured rat hippocampal neurons.

[Imaging of single molecules in live cells]. / Microscopies cellulaires à l'échelle de la molécule individuelle.

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.

Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.