Label-free detection of the material composition of nanoparticles could be enabled by the quantification of the nanoparticles' inherent dielectric response to an applied electric field. However, the sensitivity of dielectric nanoscale objects to geometric and non-local effects makes the dielectric response extremely weak. Here we show that electrostatic force microscopy with sub-piconewton resolution can resolve the dielectric constants of single dielectric nanoparticles without the need for any reference material, as well as distinguish nanoparticles that have an identical surface but different inner composition. We unambiguously identified unlabelled ~10 nm nanoparticles of similar morphology but different low-polarizable materials, and discriminated empty from DNA-containing virus capsids. Our approach should make the in situ characterization of nanoscale dielectrics and biological macromolecules possible.

Quantitative measurement of the low-frequency dielectric constants of thick insulators at the nanoscale is demonstrated utilizing ac electrostatic force microscopy combined with finite-element calculations based on a truncated cone with hemispherical apex probe geometry. The method is validated on muscovite mica, borosilicate glass, poly(ethylene naphthalate), and poly(methyl methacrylate). The dielectric constants obtained are essentially given by a nanometric volume located at the dielectric-air interface below the tip, independently of the substrate thickness, provided this is on the hundred micrometer-length scale, or larger.

We quantified the electrical polarization properties of single bacterial cells using electrostatic force microscopy. We found that the effective dielectric constant, ε(r,eff), for the four bacterial types investigated (Salmonella typhimurium, Escherchia coli, Lactobacilus sakei, and Listeria innocua) is around 3-5 under dry air conditions. Under ambient humidity, it increases to ε(r,eff) ∼ 6-7 for the Gram-negative bacterial types (S. typhimurium and E. coli) and to ε(r,eff) ∼ 15-20 for the Gram-positive ones (L. sakei and L. innocua). We show that the measured effective dielectric constants can be consistently interpreted in terms of the electric polarization properties of the biochemical components of the bacterial cell compartments and of their hydration state. These results demonstrate the potential of electrical studies of single bacterial cells.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.Super-resolution microscopy based on three-dimensional single-molecule localization microscopy (3D-SMLM) is now well established 1,2 , and its widespread adoption has led to the development of more than 36 software packages dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching 3 . While the initial emphasis in the 3D-SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (visualization of the local universe in a micro environment, pronounced 'volume'), an immersive virtual reality (VR)-based visualization software package purposefully designed to render large 3D-SMLM datasets. It is free for academic use. vLUME enables robust visualization, segmentation, annotation and quantification of millions of fluorescence puncta from any 3D-SMLM technique. vLUME has an intuitive user interface and is compatible with all commercial gaming VR hardware (Oculus Rift/Rift S and HTC Vive/Vive Pro; Supplementary Video 1). Although other microscopy data (that is, confocal) visualization tools have previously explored VR technology using volumetric representations 4,5 , vLUME has been specifically and purposefully created for SMLM. It accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics. (A comparison with other VR and non-VR tools can be found in Supplementary Table 1.)vLUME is a point-cloud based 3D-SMLM data visualization tool able to render all pointillism-based multidimensional datasets. It differs from other 3D tools for 3D-SMLM visualization such as ViSP 6 by providing a complete VR interactive environment and intuitive interface for life scientists, dedicated to data visualization, segmentation and analysis. Users load multidimensional particle-list datasets into vLUME (.csv files; Fig. 1a), such as those generated by commonly used 3D-SMLM software 7,8 . This allows users to comprehend the spatial and temporal relation between

We analyze by means of finite-element numerical calculations the polarization force between a sharp conducting tip and a non-spherical uncharged dielectric nanoparticle with the objective of quantifying its dielectric constant from electrostatic force microscopy (EFM) measurements. We show that for an oblate spheroid nanoparticle of given height the strength of the polarization force acting on the tip depends linearly on the eccentricity, e, of the nanoparticle in the small eccentricity and low dielectric constant regimes (1 < e < 2 and 1 < ε(r) < 10), while for higher eccentricities (e > 2) the dependence is sub-linear and finally becomes independent of e for very large eccentricities (e > 30). These results imply that a precise account of the nanoparticle shape is required to quantify EFM data and obtain the dielectric constants of non-spherical dielectric nanoparticles. Experimental results obtained on polystyrene, silicon dioxide and aluminum oxide nanoparticles and on single viruses are used to illustrate the main findings.

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established 1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching 3 . While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, highfidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced 'volume') a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.