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Current methods for determining equilibrium constants often operate in three-dimensional environments, which may not accurately reflect interactions with membrane-bound proteins. With our technique, based on single-molecule localization microscopy (SMLM), we directly determine protein-protein association ( K a ) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.K d ) constants in cellular environments by quantifying associated and isolated molecules and their interaction area. We introduce Kernel Surface Density (ks-density,) a novel method for determining the accessible area for interacting molecules, eliminating the need for user-defined parameters. Simulation studies validate our method's accuracy across various density and affinity conditions. Applying this technique to T cell signaling proteins, we determine the 2D association constant of T cell receptors (TCRs) in resting cells and the pseudo-3D dissociation constant of pZAP70 molecules from phosphorylated intracellular tyrosine-based activation motifs on the TCR-CD3 complex. We address challenges of multiple detection and molecular labeling efficiency. This method enhances our understanding of protein interactions in cellular environments, advancing our knowledge of complex biological processes.

Objective: To illustrate the clinical features and management of pneumatocoele presenting with a trap-valve mechanism after cochlear implantation with lateral petrosectomy (LP) in comparison with literature., Introduction: Pneumatocoele is a rare complication of cochlear implant (CI) surgery, generally managed with conservative treatments., Methods: We describe a progressively increasing pneumatocoele with a trap-valve mechanism occurring one year after CI with LP, successfully managed with revision surgery. Literature review was performed on this topic., Results: Our case was the second ever reported in literature. It appeared consequently to forceful nose-blowing in a patient with refractory nasal polyposis. Unlike the pneumatocoeles reported after standard CI, we advised revision surgery as well as in the case of pneumatocoele after staged LP and CI. In both the patients subcutaneous air collection occurred as a late complication and an insufficient Eustachian tube closure was disclosed, which is considered a complication of LP itself. Surgical management was necessary in consideration of the risk of ascending infections associated with Eustachian tube patency in LP with CI., Conclusion: We consider surgical treatment mandatory in pneumatocoele after CI with LP, it is indeed the sentinel of the restoration of Eustachian tube patency and the potential prelude to major complications.

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.

Pancreatic ductal adenocarcinoma (PDAC) continues to show no improvement in survival rates. One aspect of PDAC is elevated ATP levels, pointing to the purinergic axis as a potential attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, this axis plays essential roles in fibrosis, inflammation response, and immune function. Analyzing the main members of the PDAC extracellular purinome using publicly available databases discerned which members may impact patient survival. P2RY2 presents as the purinergic gene with the strongest association with hypoxia, the highest cancer cell-specific expression, and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y 2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification and pharmacological strategies, we demonstrate mechanistically that this ATP-driven invasion requires direct protein-protein interactions between P2Y 2 and αV integrins. DNA-PAINT super-resolution fluorescence microscopy reveals that P2Y 2 regulates the amount and distribution of integrin αV in the plasma membrane. Moreover, receptor-integrin interactions were required for effective downstream signaling, leading to cancer cell invasion. This work elucidates a novel GPCR-integrin interaction in cancer invasion, highlighting its potential for therapeutic targeting., Competing Interests: ET, MJ, QW, EC, NR, JG, AS, HK, SS, PM, RG No competing interests declared, (© 2023, Tomas Bort et al.)

Changes in ocean heat content (OHC), salinity, and stratification provide critical indicators for changes in Earth's energy and water cycles. These cycles have been profoundly altered due to the emission of greenhouse gasses and other anthropogenic substances by human activities, driving pervasive changes in Earth's climate system. In 2022, the world's oceans, as given by OHC, were again the hottest in the historical record and exceeded the previous 2021 record maximum. According to IAP/CAS data, the 0-2000 m OHC in 2022 exceeded that of 2021 by 10.9 ± 8.3 ZJ (1 Zetta Joules = 10 21 Joules); and according to NCEI/NOAA data, by 9.1 ± 8.7 ZJ. Among seven regions, four basins (the North Pacific, North Atlantic, the Mediterranean Sea, and southern oceans) recorded their highest OHC since the 1950s. The salinity-contrast index, a quantification of the "salty gets saltier-fresh gets fresher" pattern, also reached its highest level on record in 2022, implying continued amplification of the global hydrological cycle. Regional OHC and salinity changes in 2022 were dominated by a strong La Niña event. Global upper-ocean stratification continued its increasing trend and was among the top seven in 2022., (© The Author(s) 2023.)

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

The increased concentration of greenhouse gases in the atmosphere from human activities traps heat within the climate system and increases ocean heat content (OHC). Here, we provide the first analysis of recent OHC changes through 2021 from two international groups. The world ocean, in 2021, was the hottest ever recorded by humans, and the 2021 annual OHC value is even higher than last year's record value by 14 ± 11 ZJ (1 zetta J = 10 21 J) using the IAP/CAS dataset and by 16 ± 10 ZJ using NCEI/NOAA dataset. The long-term ocean warming is larger in the Atlantic and Southern Oceans than in other regions and is mainly attributed, via climate model simulations, to an increase in anthropogenic greenhouse gas concentrations. The year-to-year variation of OHC is primarily tied to the El Niño-Southern Oscillation (ENSO). In the seven maritime domains of the Indian, Tropical Atlantic, North Atlantic, Northwest Pacific, North Pacific, Southern oceans, and the Mediterranean Sea, robust warming is observed but with distinct inter-annual to decadal variability. Four out of seven domains showed record-high heat content in 2021. The anomalous global and regional ocean warming established in this study should be incorporated into climate risk assessments, adaptation, and mitigation., (© The Author(s) 2022.)

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y 2 ), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y 2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y 2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y 2 mechanistic action and therapeutic targeting.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points. Unexpectedly, these clusters do not co-localize with PAG at the membrane but instead provide a ready pool of monomers to downregulate signaling. By generating CRISPR-Cas9 knockout T cells, our data also identify that a major risk factor for autoimmune diseases, the protein tyrosine phosphatase non-receptor type 22 (PTPN22) locus, is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Quantifying the extent to which points are clustered in single-molecule localization microscopy data is vital to understanding the spatial relationships between molecules in the underlying sample. Many existing computational approaches are limited in their ability to process large-scale data sets, to deal effectively with sample heterogeneity, or require subjective user-defined analysis parameters. Here, we develop a supervised machine-learning approach to cluster analysis which is fast and accurate. Trained on a variety of simulated clustered data, the neural network can classify millions of points from a typical single-molecule localization microscopy data set, with the potential to include additional classifiers to describe different subtypes of clusters. The output can be further refined for the measurement of cluster area, shape, and point-density. We demonstrate this approach on simulated data and experimental data of the kinase Csk and the adaptor PAG in primary human T cell immunological synapses.

Plasmon excitation in metal nanoparticles triggers the generation of highly energetic charge carriers that, when properly manipulated and exploited, can mediate chemical reactions. Single-particle techniques are key to unearthing the underlying mechanisms of hot-carrier generation, transport and injection, as well as to disentangling the role of the temperature increase and the enhanced near-field at the nanoparticle-molecule interface. Gaining nanoscopic insight into these processes and their interplay could aid in the rational design of plasmonic photocatalysts. Here, we present three different approaches to monitor hot-carrier reactivity at the single-particle level. We use a combination of dark-field microscopy and photoelectrochemistry to track a hot-hole driven reaction on a single Au nanoparticle. We image hot-electron reactivity with sub-particle spatial resolution using nanoscopy techniques. Finally, we push the limits by looking for a hot-electron induced chemical reaction that generates a fluorescent product, which should enable imaging plasmonic photocatalysis at the single-particle and single-molecule levels.

The inhibition of radiative losses in dark plasmon modes allows storing electromagnetic energy more efficiently than in far-field excitable bright-plasmon modes. As such, processes benefiting from the enhanced absorption of light in plasmonic materials could also take profit of dark plasmon modes to boost and control nanoscale energy collection, storage, and transfer. We experimentally probe this process by imaging with nanoscale precision the hot-electron driven desorption of thiolated molecules from the surface of gold Fano nanostructures, investigating the effect of wavelength and polarization of the incident light. Spatially resolved absorption maps allow us to show the contribution of each element of the nanoantenna in the hot-electron driven process and their interplay in exciting a dark plasmon mode. Plasmon-mode engineering allows control of nanoscale reactivity and offers a route to further enhance and manipulate hot-electron driven chemical reactions and energy-conversion and transfer at the nanoscale.

Self-assembly processes allow designing and creating complex nanostructures using molecules as building blocks and surfaces as scaffolds. This autonomous driven construction is possible due to a complex thermodynamic balance of molecule-surface interactions. As such, nanoscale guidance and control over this process is hard to achieve. Here we use the highly localized light-to-chemical-energy conversion of plasmonic materials to spatially cleave Au-S bonds on predetermined locations within a single nanoparticle, enabling a high degree of control over this archetypal system for molecular self-assembly. Our method offers nanoscale precision and high-throughput light-induced tailoring of the surface chemistry of individual and packed nanosized metallic structures by simply varying wavelength and polarization of the incident light. Assisted by single-molecule super-resolution fluorescence microscopy, we image, quantify, and shed light onto the plasmon-induced desorption mechanism. Our results point toward localized distribution of hot electrons, contrary to uniformly distributed lattice heating, as the mechanism inducing Au-S bond breaking. We demonstrate that plasmon-induced photodesorption enables subdiffraction and even subparticle multiplexing. Finally, we explore possible routes to further exploit these concepts for the selective positioning of nanomaterials and the sorting and purification of colloidal nanoparticles.

In the discussion of resolution in optical microscopy, axial precision has often come second to its lateral counterpart. However, biological systems make no special arrangements for our preferred direction of imaging. The ability to measure axial distances, that is, the heights of fluorophores relative to a plane of reference, is thus of paramount importance and has been the subject of several recent advances. A novel method is to modify the fluorescence emission based on the height of the individual fluorophore, such that its z-position is encoded somehow in the detected signal. One such approach is metal-enhanced energy transfer, recently extended to multicolor distance measurements and applied to study the topography of the nuclear membrane. Here, the fluorescence lifetime is shortened due to the proximity of the fluorophores to a thin metallic surface. Fluorescence lifetime imaging can therefore be used as an axial ruler with nanometer precision.

DNA origami is a powerful approach for assembling plasmonic nanoparticle dimers and Raman dyes with high yields and excellent positioning control. Here we show how optothermal-induced shrinking of a DNA origami template can be employed to control the gap sizes between two 40 nm gold nanoparticles in a range from 1 to 2 nm. The high field confinement achieved with this optothermal approach was demonstrated by detection of surface-enhanced Raman spectroscopy (SERS) signals from single molecules that are precisely placed within the DNA origami template that spans the nanoparticle gap. By comparing the SERS intensity with respect to the field enhancement in the plasmonic hot-spot region, we found good agreement between measurement and theory. Our straightforward approach for the fabrication of addressable plasmonic nanosensors by DNA origami demonstrates a path toward future sensing applications with single-molecule resolution.

A Au-silica Janus particle is elevated along the laser beam axis in an optical trap. The propulsion mechanism is based on the local temperature gradient created around the particle due to the photothermal conversion of the gold-coated hemisphere. The height of the particle and its motion-direction are tuned by the nature and the concentration of the electrolytes in the medium., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Visible-light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single-molecule study of the plasmon-assisted isomerization of a photochrome-fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser. The metallic nanostructures significantly enhance the visible light-induced performance of the photoconversion, which normally requires ultraviolet excitation. The resulting ring-open isomer is strongly fluorescent and can also be excited at 633 nm. These stochastic emission events are used to monitor photochromic activation and show quadratic dependence on incident power. The utilization of a single laser wavelength for both photochromic activation and excitation effectively mimics a pseudo two-colours system., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, we use the photoisomerization of azobenzenes, a phenanthrospirooxazine, and a fulgide in a twisted nematic liquid crystalline phase to change the chiral twisting power of the system. The changes are probed by the rotatory power of linearly polarized light. Time resolved and steady state experiments are carried out. The chiral change and the photoisomerization process have similar characteristic recovery times and activation energy, thus probing that the change is induced by the modification in the chemical composition of the photochromic dopant system. The amplitude of the light twisting power change correlates with the order change in the liquid crystal (LC) but not with the modification in the absorption characteristics of the system. This indicates that the driving force of the chiral change is the microscopic order modification in the LC phase that affects the helical pitch of the phase., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Plasmon excitation of spherical gold nanoparticles carrying a fluorescent labeled 30 bp dsDNA cargo, with one chain covalently attached through two S-Au bonds to the surface, results in release of the complementary strand as ssDNA that can be examined in situ using high-resolution fluorescence microscopy. The release is dependent on the total energy delivered, but not the rate of delivery, an important property for plasmonic applications in medicine, sensors, and plasmon-induced PCR.