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Updated: Sep 30, 2025

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Simultaneous Multicolor DNA-PAINT without Sequential Fluid Exchange Using Spectral Demixing.

Niclas Gimber¹, Sebastian Strauss^2,3, Ralf Jungmann^2,3

¹Advanced Medical Bioimaging Core Facility, Charité-Universitätsmedizin, 10117 Berlin, Germany.

|March 15, 2022

Summary

This summary is machine-generated.

We developed simultaneous multicolor spectral demixing DNA-PAINT (SD-DNA-PAINT) for faster nanoscale imaging. This method avoids fluid exchange, enabling rapid, high-precision multicolor imaging of biological structures.

Keywords:

DNA-PAINT Super-resolution fluorescence microscopy multicolor single-molecule imaging single-molecule localization microscopy spectral demixing

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Area of Science:

Biophysics
Microscopy
Nanotechnology

Background:

Multicolor single-molecule localization microscopy (SMLM) is crucial for understanding nanoscale biological structures.
DNA-PAINT offers high localization precision but multicolor variants often require slow sequential imaging.
Exchange-PAINT, a common multicolor DNA-PAINT method, necessitates fluid exchange, increasing acquisition time.

Purpose of the Study:

To develop a faster, simultaneous multicolor DNA-PAINT method.
To overcome the limitations of sequential imaging in Exchange-PAINT.
To enable efficient and precise multicolor nanoscale imaging.

Main Methods:

Combined DNA-PAINT with spectral demixing (SD) for simultaneous multicolor acquisition.
Developed novel probes and a multichannel registration procedure.
Utilized spectral demixing (SD) to differentiate simultaneously imaged colors with minimal crosstalk.

Main Results:

Achieved simultaneous multicolor SD-DNA-PAINT with high localization precision (3-6 nm).
Demonstrated minimal crosstalk between simultaneously imaged channels.
Successfully registered dual- and triple-color imaging on DNA origami and cellular structures.

Conclusions:

Simultaneous multicolor SD-DNA-PAINT significantly accelerates multicolor imaging acquisition.
The novel approach maintains high localization precision and registration accuracy.
This method provides a powerful tool for nanoscale biological imaging without fluid exchange.