More than just location- expanding the use of PAINT for multi-dimensional imaging

Super-resolution techniques allow biological systems to be studied at the nanoscale, but are usually restricted to providing positional information only. Point Accumulation for Imaging in Nanoscale Topography (PAINT) makes use of diffusing probes that are localised as they bind to a surface. We have further developed this technique to provide further information on the environment of the fluorophore. This was achieved by adapting a total internal reflection fluorescence microscope by inserting a diffraction grating in the image plane, allowing both the position and wavelength of single emitters to be determined simultaneously. We have used this with a probe whose wavelength varies depending on its environment to nanoscopically map the hydrophobicity of liposomes, amyloid aggregates and cells. Although demonstrated with a hydrophobicity probe, this technique is applicable to many other wavelength shifting dyes.