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.

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 pre-determined 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 nano-sized 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 towards localized distribution of hot electrons, contrary to uniformly distributed lattice heating, as the mechanism inducing Au-S bond breaking. We demonstrate that plasmon-induced photo-desorption enables sub-diffraction and even sub-particle 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.

Several forms of niobium oxide were prepared, including nanostructured mesoporous materials, and their acidity properties were comprehensively investigated and compared with commercially available materials. The composites were characterized by a variety of techniques, including XRD, TEM, N₂ adsorption and Hammett acid indicator studies. The acidity of the niobium oxide derivatives was also investigated by the ability of the materials to successfully promote the halochromic ring-opening of an oxazine-coumarin probe that was specifically designed for use in fluorescence imaging studies. The ring-opening reaction was easily monitored using UV-visible, fluorescence and NMR spectroscopy. Single molecule microscopy was employed to gain a more in-depth understanding of the niobium oxide acid catalysis pathway. Using this technique, the rate of niobium oxide mediated protonation was estimated to be 1.8 × 10⁻¹³ mol m⁻² s⁻¹. Single molecule analysis was also used to obtain a detailed map of Brønsted acid sites on the niobium oxide surface. The active sites, located by multiple blinking events, do not seem to be localized on any area of the material, but rather randomly distributed throughout the solid state surface. As the reaction proceeds, the sites with the highest acidity and accessibility are gradually consumed, making the next tier of acid sites available for reaction. The phenomenon was more closely characterized by using time lapsed reactivity maps.

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.

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.