High-density volumetric super-resolution microscopy.

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs µm-2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs µm-2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs µm-2) in dense cytosolic tubulin datasets.

Recyclable thermosets based on modified epoxy-amine network polymers.

The development of high performance, recyclable thermoset materials for applications in plastics, composites, coatings and adhesives requires a synthetic approach where recyclability is designed into the molecular structure of the material. This paper describes a single stage process for the creation of materials from simple, low-cost molecular building blocks, where the polymerisation of liquid epoxy resins and aliphatic amines in the presence of an n-butyl diboronic ester, delivers epoxy-amine-dioxazaborocane materials with tunable physical properties including glass transition temperature (Tg). Mechanical (thermal) recycling and reprocessing of the epoxy-amine-dioxazaborocane thermoset is demonstrated, with retention of Young's modulus and ultimate tensile strength. Most notably, an efficient and low-cost process for the chemical recycling, disassembly and dissolution of the thermoset is demonstrated via two complementary processes using either pinacol (diol) or mono-functional phenylboronic ester.

resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.