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Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy.

Karoline Friedl1, Adrien Mau2, Fanny Boroni-Rueda3

  • 1Aix Marseille Université, CNRS, INP UMR7051, NeuroCyto, 13005 Marseille, France; Abbelight, 191 Avenue Aristide Briand, 94230 Cachan, France.

Cell Reports Methods
|September 26, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces faster multicolor single-molecule localization microscopy (SMLM) methods. Spectral demixing in dSTORM (SD-dSTORM) offers a robust option for advanced multicolor SMLM investigations.

Keywords:
CP: ImagingDNA-PAINTSMLMSTORMcrosstalkdSTORMfluorescencemulticolormultitargetspectral demixingsuper resolution microscopy

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Nanotechnology

Background:

  • Single-molecule localization microscopy (SMLM) achieves nanoscale resolution.
  • Techniques like STORM and DNA-PAINT are established SMLM methods.
  • Faster multicolor SMLM is crucial for complex biological imaging.

Purpose of the Study:

  • To implement and evaluate two novel approaches for faster multicolor SMLM.
  • To assess the crosstalk and biological relevance of spectral demixing in dSTORM (SD-dSTORM).
  • To extend these methods to 3D and three-color imaging.

Main Methods:

  • Developed simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) and spectral demixing dSTORM (SD-dSTORM).
  • Split emitted fluorescence between two cameras for simultaneous imaging.
  • Evaluated SD-dSTORM crosstalk using DNA nanorulers and labeled cells.
  • Assessed crosstalk impact on detecting subdiffraction patterns.

Main Results:

  • S2C-DNA-PAINT served as a reference for low crosstalk.
  • SD-dSTORM crosstalk was evaluated across various sample types.
  • Crosstalk effects on biological patterns were assessed.
  • Successful extension to 3D acquisition and three-color imaging was demonstrated.

Conclusions:

  • Spectral demixing (SD-dSTORM) is a viable and versatile method for multicolor SMLM.
  • These advancements enable more robust investigations in nanoscale imaging.
  • The developed techniques enhance the capabilities of SMLM for biological research.