High-Throughput Hyperspectral and Multiplexed Super-Resolution Fluorescence Imaging by SP-STORM.

Simultaneous determination of spatial location and spectral color of single molecules at large molecular density with high throughput was achieved by combining single-molecule photoswitching and optical in-hardware Fourier transformation of single-molecule emission spectra into the phasor space. The method, named as spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM), achieved simultaneous super-resolution imaging of five subcellular structures in parallel with minimum crosstalk for the first time. The high-throughput feature of SP-STORM enables these subcellular structures to be readily resolved in about one min, which is more than an order of magnitude faster than other multiplexing single-molecule localization microscopy techniques. The concept of SP-STORM is also compatible with and can be readily applicable to other super-resolution microscopy.