Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities

Koen J.A. Martens*, Martijn Gobes, Emmanouil Archontakis, Roger R. Brillas, Niels Zijlstra, Lorenzo Albertazzi, Johannes Hohlbein*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

3 Citations (Scopus)


Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0thand 1storder provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Original languageEnglish
Pages (from-to)8618-8625
Number of pages8
JournalNano Letters
Issue number21
Publication statusPublished - 9 Nov 2022


  • multicolor imaging
  • point accumulation for imaging in nanoscale topography (PAINT)
  • single-molecule Förster resonance energy transfer (smFRET)
  • Single-molecule spectroscopy
  • stochastic optical reconstruction microscopy (STORM)


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