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Updated: Jan 13, 2026

Robust 3D DNA FISH Using Directly Labeled Probes
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Optimizing Effective Labeling Efficiency in MINFLUX 3D DNA-PAINT Microscopy by Maximizing Marker Detection

Christian Soeller1, Alexandre F E Bokhobza1, Javier Casares-Arias2

  • 1Department of Physiology, University of Bern, 3012 Bern, Switzerland.

ACS Photonics
|January 12, 2026
PubMed
Summary
This summary is machine-generated.

We optimized single-molecule localization microscopy (MINFLUX) by improving labeling efficiency (ELE) in 3D DNA-PAINT. Repeat DNA-PAINT significantly reduced site-loss, increasing marker detection for higher resolution imaging.

Keywords:
DNA-PAINTMINFLUX nanoscopydocking-site stabilityeffective labeling efficiencymarker detectionsuper-resolution microscopy

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Area of Science:

  • Biophysics
  • Super-resolution microscopy
  • Molecular imaging

Background:

  • MINFLUX offers high 3D resolution but lacks clear acquisition termination criteria.
  • Current methods face limitations in effective labeling efficiency (ELE) over time.

Purpose of the Study:

  • Systematically investigate ELE and saturation in 3D MINFLUX DNA-PAINT.
  • Optimize imaging parameters for Nup96 proteins in U-2 OS cells.
  • Enhance marker detection probability for improved resolution.

Main Methods:

  • Utilized a commercial MINFLUX microscope for 3D DNA-PAINT imaging.
  • Quantitatively measured ELE using maximum-likelihood template fitting.
  • Compared conventional DNA-PAINT with repeat DNA-PAINT.

Main Results:

  • Achieved ~60% ELE, often requiring hours of acquisition.
  • Identified docking strand site-loss as a key limitation in conventional DNA-PAINT.
  • Repeat DNA-PAINT reduced site-loss, increasing site visits >3-fold.
  • Repeat DNA-PAINT enabled ELE to reach chemical labeling limits.

Conclusions:

  • Maximizing marker detection is crucial for reaching MINFLUX resolution limits.
  • Repeat DNA-PAINT overcomes site-loss, enhancing labeling efficiency.
  • Optimized ELE via repeat DNA-PAINT improves 3D super-resolution imaging capabilities.