Spatially resolved refractometry, fluorophore-concentration, axial-position, and orientational imaging using an evanescent Bessel beam

Simultaneous field- and aperture-plane (back-focal plane, BFP) imaging enriches the information content of fluorescence microscopy. In addition to the usual density and concentration maps of sample-plane images, BFP images provide information on the surface proximity and orientation of molecular fluorophores. They also give access to the refractive index of the fluorophore-embedding medium. However, in the high-NA, wide-field detection geometry commonly used in single-molecule localisation microscopies, such measurements are averaged over all fluorophores present in the objective's field of view, thus limiting spatial resolution and specificity. We here solve this problem and demonstrate how an oblique, variable-angle, coherent ring illumination can be used to generate a Bessel beam that - for supercritical excitation angles - produces an evanescent needle of light. Scanning the sample through the this evanescent needle enables us to acquire combined sample-plane and BFP images with sub-diffraction resolution and axial localisation precision. Background, resolution and polarisation considerations will be discussed.