Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

Abstract

Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…This phase separation remains challenging to image with other techniques 56 . Spectrally-resolved PAINT (sPAINT) has also been introduced 57 In addition to the characterization of morphology and size, insight on the internal structure of micelles and vesicles provides valuable information. A first quantitative study to assess encapsulation and compartmentalization in nanostructured lipid carriers (NLC) was performed by Boreham et al 59 .…”

Single-molecule 3D orientation imaging reveals nanoscale compositional heterogeneity in lipid membranes

“…13 However, the order parameter is an ensemble average taken and cannot unambiguously determine the 3D orientation of a single molecule (SM). 14 Spectrally-resolved SM localization microscopy (SMLM) [15][16][17] maps the local polarity or hydrophobicity of protein aggregates and subcellular structures, 17,18 and fluorescence lifetime imaging identifies subresolution lipid domains in the plasma membrane. 19 However, these approaches require specific environment-sensitive fluorescent probes (e.g., Nile red, 20 Laurdan, 21 and 3-hydroxyflavone derivatives 22 ) whose fluorescence spectra (intensities) or lifetimes are sensitive to local environment.…”

Optical Structural Analysis of Individual α‐Synuclein Oligomers

“…We used a single‐molecule sensitive TIRF imaging mode to measure the structural arrangement of individual spatially isolated diffraction‐limited aggregates which we have previously characterised using super‐resolution techniques 23. We performed an aggregation reaction for recombinant α‐synuclein and focused on the kinetics of the lag phase of the aggregation 24.…”

“…We used as ingle-molecule sensitive TIRF imaging mode to measure the structural arrangement of individual spatially isolated diffraction-limited aggregates which we have previously characterised using super-resolution techniques. [23] We performed an aggregation reaction for recombinant a-synuclein and focused on the kinetics of the lag phase of the aggregation. [24] Ther eaction was done at ac oncentration of 70 mm under agitation at 200 rpm in 25 mm Tris buffer (pH 7.4) supplemented with 0.1m NaCl and 0.01 %N aN 3 at 37 8 8C.…”