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 in-hardware transformation based spectral phasor analysis. The method, named spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM), achieved simultaneous super-resolution imaging of five subcellular structures 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 minute, which is more than a 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.