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Super-resolution Fluorescence Microscopy

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...

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Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Published on: January 6, 2026

Super-resolution microscopy with DNA-PAINT.

Joerg Schnitzbauer^1,2, Maximilian T Strauss^1,2, Thomas Schlichthaerle^1,2

¹Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.

Nature Protocols

|May 19, 2017

Summary

This summary is machine-generated.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) provides a simple super-resolution microscopy method using DNA probes. This technique achieves molecular-scale spatial resolution and enables spectrally unlimited multiplexing for biological research.

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Area of Science:

Biophysics
Molecular Biology
Microscopy

Background:

Super-resolution microscopy overcomes the diffraction limit of light for biological imaging.
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a localization-based super-resolution technique utilizing DNA probes.
Current super-resolution methods face limitations in dye photophysics and multiplexing capabilities.

Purpose of the Study:

To present a comprehensive protocol for the DNA-PAINT framework.
To enable both novice and expert users to implement DNA-PAINT for super-resolution imaging.
To detail the computational and experimental aspects of DNA-PAINT, including data processing and analysis.

Main Methods:

Utilizing transient binding of dye-labeled imager oligonucleotides to complementary DNA docking strands for stochastic blinking.
Developing DNA origami test samples for protocol validation.
Implementing multiplexed data acquisition, simulation, and super-resolution image reconstruction.
Employing post-processing techniques like drift correction, quantitative molecule counting (qPAINT), and particle averaging.
Providing the integrated software package 'Picasso' for computational analysis.

Main Results:

Achieved molecular-scale spatial resolution (sub-5-nm) with approximately 1-nm localization precision.
Enabled spectrally unlimited multiplexing for advanced imaging applications.
Demonstrated precise molecule counting capabilities.
The protocol is modular and adaptable for various in vitro and cellular applications.

Conclusions:

DNA-PAINT offers a versatile and powerful super-resolution microscopy technique.
The presented protocol and software facilitate the implementation and application of DNA-PAINT.
This method overcomes limitations of existing super-resolution techniques, expanding possibilities in biological research.