Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

We have developed a multi-dimensional super-resolution (md-SR) imaging technique to determine both the localization and the environmental properties of single-molecule fluorescent emitters. The method, termed sPAINT, exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule to enable the mapping of hydrophobicity of biological structures. We first validated the sPAINT method by studying synthetic lipid vesicles of known composition, then applied it to measure the hydrophobicity of amyloid fibrils and oligomers implicated in neurodegenerative diseases, and of the plasma membrane of mammalian cells. sPAINT is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, which enables all the necessary information to be extracted simultaneously from a single image plane. sPAINT enables the hydrophobicity of surfaces to be mapped at the nanoscale in a dynamic fashion.Medical Research Council (Grant ID: MR/K015850/1), Engineering and Physical Sciences Research Council, Royal Society (University Research Fellowship, Grant ID: UF120277), Augustus Newman Foundation, Cambridge Advanced Imaging Centre, Christ’s Colleg

Ultrasensitive Measurement of Ca(2+) Influx into Lipid Vesicles Induced by Protein Aggregates.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca(2+) entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca(2+) sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca(2+) influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability

Research data supporting "Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping"

Supplementary video for data analysis.MRC [MR/K015850/1] Wellcome Trust [UF120277