Figure 1.

A bottom-up approach toward multidimensional super-resolution microscopy. (a) Emitting single molecules are kept at a low density in the wide field, so that they could be each independently evaluated for their nanoscale positions and high-dimensional properties. (b) Examples of multidimensional single-molecule observables that may be encoded-decoded, including localization in 3D, color identity, spectrum, fluorescence polarization, motion and diffusion, and fluorescence lifetime. (c) Stochastic sampling of single molecules over many camera frames, e.g., through photoswitching or diffusional exchange, to enable accumulation of the single-molecule quanta. (d) The accumulated single-molecule measurements enable local statistics to extract meaningful parameters at the nanoscale. (e) Resultant multidimensional super-resolution images, with the possibility to integrate measurements of different dimensions. The palette scheme in (b) is adapted from ref¹²⁰.