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We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

As one of the countries in the world with the highest growth of internet users, Indonesia is experiencing a rapid growth in social media usage . Some use social media for networking but some others use it to spread hoaxes, fake information, or disinformation. During presidential election in Indonesia in the period from 2017 to 2019, hoaxes and disinformation were widely circulated through social media and instant messaging. This phenomenon has triggered heated public debates on the nexus between digital spaces and security, which include how the online disinformation has threatened Indonesian security. For example , hoaxes were represented in the public sphere as an existential threat to Indonesi an unity . Immediate question regarding this phenomenon is: why are online hoaxes and fake information represented in public spheres as a security threat? This paper argues that as a response toward the increase of online hoaxes, there were securitizing moves made by political elites and special agencies in Indonesia before and in the aftermath of the 2019 Indonesian presidential election. Employing discourse analysis of selected relevant news articles around the period of 2017-2019, this paper analyses the dynamic of the securitization of online hoaxes in Indonesia. Grounded within Securitization Theory, this paper analyses; the facilitating condition ; the triumvirates of securitizations, which are the securitizing actors, the threats posed by hoaxes, and the audiences; as well as extraordinary measures executed to handle the threat s; internet throttling and internet shutdown when necessary.

We provide evidence of a local synaptic nano-environment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nano-environment might play a role in the regulation of brain activity.

Measuring carbon nanotube diffusion is complex in 3D liquid environments. Single molecule fluorescence microscopy commonly provides nanotube trajectories in the 2D imaging plane with nanometer precisions but assessing the third dimension is more challenging task. To this aim, We will present two strategies based on point-spread function (PSF) engineering [1] or self-interfering PSF [2]. Because nanotubes are not spherical objects, 3D angular diffusion of the nanotubes shall also be considered. We will show that using a high-frame rate imaging (kHz) of nanotube movements, the autocorrelation time of nanotube fluorescence intensity can be computed in order to measure the rotational diffusion coefficient of the nanotubes. This further allows to estimate the length of the nanotubes either from the rotational diffusion coefficients alone, or by combining translational and rotational diffusion coefficients which has the advantage to avoid the requirement of knowing the solution viscosity or the SWCNT hydrodynamic diameter [3]. References [1] Gresil, Lee, et al. In preparation [2] Caceido, Lee, et al. In preparation [3] Lee & Cognet J. Appl. Phys. 128 (2020) 224301

In 1932 a deer antler carved into the form of an approximately life-sized, three-dimensional, erect human phallus was discovered alongside Romano-British activity at Colsterworth Quarry, Lincolnshire and donated to Grantham Museum. Never previously subject to a discussion in print, this article considers the phallus through the lenses of apotropaic magic and the magico-medicinal and socio-religious significance of deer and deer products in Roman Britain. The original context of discovery is not well recorded, though likely relates to an area of industrial activity with an associated settlement nearby. A variety of potential functional and ritual contexts for the antler phallus are considered: as a fragment of religious statuary, an apotropaic device on a building or vehicle, a votive offering, and as part of a tool or vessel. These discussions explore the highly contextualised applications of embodied and disembodied phallic imagery in Roman Britain, the liminal space between concepts of religion and magic, and the significance of materiality and embodied interaction when considering the socio-religious significance of phallic imagery.

International audience; In localization microscopy, the position of isolated fluorescent emitters are estimated with a resolution better than the diffraction limit. In order to image thick samples, which are common in biological applications, there is considerable interest in extending the depth-of-field of such microscopes in order to make their accuracy as invariant as possible to defocus. For that purpose, we propose to optimize annular binary phase masks placed in the pupil of the microscope in order to generate a point spread function for which the localization accuracy is almost invariant along the optical axis. The optimization criterion is defined as the localization accuracy in the plane expressed in terms of the Cramér-Rao bound. We show that the optimal masks significantly increase the depth-of-field of single-molecule imaging techniques relatively to an usual microscope objective.

International audience; Single-molecule localization microscopy has become a prominent approach to study structural and dynamic arrangements of nanometric objects well beyond the diffraction limit. To maximize localization precision, high numerical aperture objectives must be used; however, this inherently strongly limits the depth-of-field (DoF) of the microscope images. In this work, we present a framework inspired by "optical co-design" to optimize and benchmark phase masks, which, when placed in the exit pupil of the microscope objective, can extend the DoF in the realistic context of single fluorescent molecule detection. Using the Cramér-Rao bound (CRB) on localization accuracy as a criterion, we optimize annular binary phase masks for various DoF ranges, compare them to Incoherently Partitioned Pupil masks and show that they significantly extend the DoF of single-molecule localization microscopes. In particular we propose different designs including a simple and easy-to-realize two-ring binary mask to extend the DoF. Moreover, we demonstrate that a simple maximum likelihood-based localization algorithm can reach the localization accuracy predicted by the CRB.The framework developed in this paper is based on an explicit and general information theoretic criterion, and can thus be used as an engineering tool to optimize and compare any type of DoF-enhancing phase mask in high resolution microscopy on a quantitative basis.

International audience; The brain extracellular space (ECS) is a complex network that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites 1. Signaling molecules, neuromodulators, and nutrients transit via the ECS, therefore mediating the communication between cells. Despite the relevance of this important part of the brain, its dynamics and structural organization at the nanoscale is still mostly unknown 2. We have recently demonstrated that single-walled carbon nanotubes (SWCNTs) can be used to image and probe live brain tissue, providing super-resolved maps of the brain ECS and quantitative information on the local diffusion environment 3,4. Here, we propose an important refinement of this approach by implementing a structured illumination technique (named HiLo microscopy 5) to image fluorescently labelled neuronal structures in parallel to SWCNT NIR imaging. This technique is based on speckle illumination and relies on the acquisition of one structured and one uniform illumination image to obtain images deep into tissues with good optical sectioning. Having access to spatially resolved SWCNT diffusivity around specific neuronal structures will provide more precise insights about the heterogeneity of the brain environment.

This paper scrutinizes two relatively similar cyber activisms in Indonesia, namely Guard the 2014 General Election and Guard the 2015 Local Election. The two movements serve as cases to study cyber activisms contributions to democracy. Guard the General Election, which received massive support from the internet users, has been acknowledged as a success story of a cyber political crowdsourcing in Indonesia. Guard the Local Election tried to repeat the success a year after, but received fewer supports. By scrutinizing those movements, this writing attempts to answer two connected questions of (1) how can cyber social movements contribute to democracy? (2) Why were some cyber movements received more popular support than the others? This paper argues, these movements have contributed to democratization in the way that the activisms reshaped civic culture; introducing new practices, empowering citizens identities, and strengthening trust. Also, the writing explores arguments that political momentum and mainstream media coverage are influential on determining the successfulness of cyber movements. Methodologically, this paper subscribes to qualitative content analysis as a tool to examine interviews materials as well as online and offline texts.

The Roman period is ubiquitous in popular media and the public consciousness, featuring regularly in formal education programmes, on film and television, and in museum displays. Scholarship into th...

Luminescent single wall carbon nanotubes are now well established, as unique nanoreporters to probe the brain extracellular space[1]. On the imaging side, this comes from their rich near-infrared optical properties, which also eventually be improved by sp3-chemical functionalization[2]. In addition, their uncommon 1D morphology is an important asset for tissue penetration, yet understanding their diffusion behavior in the complex brain extracellular network is challenging and necessitates dedicated analysis tools. To this aim, we have developed two novel approaches (i) based on the local analysis of trajectory contours to locally measure the nanoscale dimensions of the brain network [3] and (ii) based on the transient evaluation of anomalous carbon nanotube diffusion to delineate the ECS molecular diffusion landscape [4]. The application of these analytical tools to extract relevant biological parameters in physiological and pathological brain models will be presented [5]. References [1] Godin et al Nat. Nanotechnol. 12 (2017) 238-243 ; Gao, et al, Nanomaterials 7, 11, (2017) 393 ; Danné et al ACS Photonics, 5, 2, (2018) 359-364 [2] Mandal et al. Scientific Reports, 10 (2020) 5286 [3] Paviolo et al Methods 174 (2020) 91-99 [4] Lee et al, in preparation [5] Soria et al Nat. Commun., 11 (2020) 3440

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.HighlightsA single-molecule unzipping assay mimics DNA unwinding by Pol II and maps the topography of human canonical, H2A.Z and uH2B nucleosome barriers at high resolutionReal-time dynamics and full molecular trajectories of Pol II crossing the nucleosomal barrier reveal the transcriptional landscape of the barrier at high accuracyH2A.Z enhances the width and uH2B the height of the barrierA unified mechanical model links position-dependent dwell times of Pol II on the nucleosome with energetics of the barrier

International audience; A new approach is presented to measure the length distribution of dispersed single-walled carbon nanotubes (SWCNT). In this method, the diffusive trajectories of individual SWCNTs in solution are reconstructed from high-frame rate video stacks. These trajectories allow the estimation of two key statistics for the SWCNTs: their translational diffusion coefficient, and the autocorrelation time of their fluorescence intensity. We show that the autocorrelation time is a measure of the rotational diffusion coefficient of the SWCNTs, and that the length of the SWCNTs can be estimated either from the rotational diffusion coefficients alone, or by combining translational and rotational diffusion coefficients. Moreover, this last estimate does not require knowledge of the solution viscosity or of the SWCNT hydrodynamic diameter.

Significance Optical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible., In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a “time-shared” dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.

Super-resolution microscopy provides direct insight into fundamental biological processes occurring at length scales smaller than light’s diffraction limit. The analysis of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data analysis methods starting from an overview of basic statistical techniques underlying the analysis of super-resolution and, more broadly, imaging data. We subsequently break down the analysis of super-resolution data into four problems: the localization problem, the counting problem, the linking problem, and what we’ve termed the interpretation problem.

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

This paper aims to explain how and why the representations of transit forced migrants in Indonesian major print media were vastly different within two time spans: (1) during the arrivals of the Indochinese refugees in 1975-1996 and (2) in the period of the new generations of refugees from Middle Eastern and South Asian countries in 1997-2013. Employing media content analysis of 216 news articles from three major print media in Indonesia, this study found out that the Indochinese refugees were portrayed with positive labels and thus, mainly discussed in connection with the non-security theme. In contrast, the new generations of forced migrants were portrayed with negative labels such as ‘illegal immigrants’ and were framed as security threats. Grounded within Securitization Theory, this paper thus argues that the changing representations were caused by the securitizing move made by specialized agencies in Indonesia.

The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble in vivo. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from Thermotoga maritima both in vivo and in vitro by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a simple and rapid purification protocol and demonstrate the thermal and pH stability of the shell. Efforts to study the biophysical effects of protein encapsulation have been problematic in complex compartments, but the simplicity of assembling and loading encapsulin makes it an ideal system for future experiments exploring the effects of encapsulation on proteins.

We present a single molecule method for counting proteins within a diffraction-limited area when using photoactivated localization microscopy. The intrinsic blinking of photoactivatable fluorescent proteins mEos2 and Dendra2 leads to an overcounting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe blinking and show that Dendra2 photobleaches three times faster and blinks seven times less than mEos2, making Dendra2 a better photoactivated localization microscopy tag than mEos2 for molecular counting. The simultaneous activation of multiple molecules is another source of error, but it leads to molecular undercounting instead. We propose a photoactivation scheme that maximally separates the activation of different molecules, thus helping to overcome undercounting. We also present a method that quantifies the total counting error and minimizes it by balancing over- and undercounting. This unique method establishes that Dendra2 is better for counting purposes than mEos2, allowing us to count in vitro up to 200 molecules in a diffraction-limited spot with a bias smaller than 2% and an uncertainty less than 6% within 10 min. Finally, we demonstrate that this counting method can be applied to protein quantification in vivo by counting the bacterial flagellar motor protein FliM fused to Dendra2.

During transcription elongation, E. coli RNA polymerase (RNAP) alternates between periods of processive nucleotide addition and periods of pausing. Various mechanisms promote pausing. In particular, increased pausing at a “consensus pause element” of the form G-10Y-1G+1 (where Y-1 corresponds to the 3′ RNA end) has been described (Science 344:1042, 2014; Science 344:1285, 2014). Such pause sites have been shown to exhibit increased efficiency in the RNAP βD446A mutant; this effect can be understood from the interaction between WT RNAP βD446 and G+1, which stabilizes RNAP in a post-translocated register in the presence of a G+1, and thus decreases pausing (Science 344:1285, 2014).In this work, we use high-resolution optical tweezers to characterize the dynamics of both processive nucleotide elongation and pausing by WT and βD446A RNAP at very high spatial and temporal resolutions, under various force conditions. Our ability to resolve sub-second pausing events provide new insights into the dynamics of RNAP translocation and pausing during elongation, and their modulation by sequence-specific RNAP-DNA interactions.

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.

Super-resolution microscopy, a powerful technique for determining protein localization, cannot discriminate between random colocalization of proteins and actual protein-protein interactions. Bimolecular fluorescence complementation, on the other hand, directly probes inter-molecular interactions, but has been classically limited by the diffraction limit. Here, we demonstrate that three photoconvertible fluorescent proteins (PA-FP) -- PAmCherry1, Dendra2 and a Dronpa variant -- support fluorescence complementation, and that split-PA-FPs can be used after complementation for photoactivated localization microscopy (PALM). We recovered photoconvertible fluorescent complexes by fusing split-PAmCherry1 to two interacting subunits of mammalian ATP-synthase. PALM images obtained in situ from such constructs were of quality similar to those obtained by labeling and imaging a single ATP-synthase subunit; however, in this scheme, each PALM event corresponded not only to a single ATP-synthase complex localized with ∼20nm accuracy, but also, more precisely, to two subunits of that complex interacting within

International audience; The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.

We present a single molecule method for counting photoactivatable proteins, mEos2 and Dendra2, within a diffraction limited area when using photo-activated localization microscopy (PALM). The intrinsic blinking of photoactivatable fluorescent proteins (PAFPs) mEos2 and Dendra2, leads to an over-counting error, which constitutes a major obstacle for their use as molecular counting tags. Here, we introduce a kinetic model to describe the photo-blinking of Dendra2 and mEos2 proteins and use the measured blinking parameters to address the over-counting error. Simultaneous photoactivation of multiple molecules is another source of error, but it leads to molecular undercounting, instead. We propose a novel photo-activation scheme that separates uniformly the activation of different molecules, thus helping to decrease the undercounting error. Our method quantifies the total counting error and minimizes it by balancing the over- and undercounting.