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Related Experiment Video

Updated: Feb 28, 2026

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM
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High-Throughput Hyperspectral and Multiplexed Super-Resolution Fluorescence Imaging by SP-STORM.

Elric Dion Pott1, Meek Yang1, James Ethan Batey1

  • 1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States.

JACS Au
|February 27, 2026
PubMed
Summary

Spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM) achieves high-throughput, simultaneous super-resolution imaging of five subcellular structures. This novel technique rapidly determines molecular location and spectral color, overcoming previous limitations in speed and multiplexing.

Keywords:
5-plexed 3D SMLM in parallelHigh throughputHyperspectral super-resolution imagingSingle molecule spectroscopySpectral phasor

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Molecular Imaging

Background:

  • Super-resolution microscopy techniques enable visualization of subcellular structures beyond the diffraction limit.
  • Multiplexing single-molecule localization microscopy (SMLM) allows simultaneous imaging of multiple targets but often suffers from low throughput and crosstalk.
  • Accurate determination of both spatial location and spectral properties of single molecules is crucial for advanced biological imaging.

Purpose of the Study:

  • To develop a high-throughput method for simultaneous super-resolution imaging of multiple subcellular structures.
  • To combine single-molecule photoswitching with optical Fourier transformation for enhanced molecular analysis.
  • To introduce spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM) for improved multiplexed SMLM.

Main Methods:

  • Integration of single-molecule photoswitching with in-hardware optical Fourier transformation to map emission spectra into phasor space.
  • Development of spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM) for simultaneous localization and spectral unmixing of single molecules.
  • Parallel imaging of five distinct subcellular structures with minimized crosstalk.

Main Results:

  • SP-STORM successfully achieved simultaneous super-resolution imaging of five subcellular structures with minimal crosstalk.
  • The method demonstrated high throughput, resolving structures in approximately one minute.
  • This represents a significant speed improvement (over an order of magnitude) compared to existing multiplexing SMLM techniques.
  • The technique accurately determined both spatial location and spectral color of single molecules at high densities.

Conclusions:

  • SP-STORM offers a novel and efficient approach for high-throughput, multiplexed super-resolution microscopy.
  • The method overcomes key limitations of current SMLM techniques, particularly in speed and parallel imaging capabilities.
  • The underlying concept of SP-STORM is adaptable to other super-resolution microscopy platforms, broadening its potential impact.