biostudies-literaturePott EDUniversity of ArkansasSociety for Analytical Chemists of PittsburghArkansas Biosciences Institute788-794https://www.ebi.ac.uk/biostudies/studies/S-EPMC12933340biostudies-literatureUnknown6(2)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 isJACS AuHigh-Throughput Hyperspectral and Multiplexed Super-Resolution Fluorescence Imaging by SP-STORM.PMC12933340PG105140Batey JEPott EDYang MEmbree JDong BfalseHigh-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 is2026-01-01T00:00:00Z2026 Feb2026-07-16T22:11:23.075Z2026-07-14T03:03:42.233ZS-EPMC129333404175584910.1021/jacsau.5c01677