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High-Throughput Single-Molecule Microscopy with Adaptable Spatial Resolution Using Exchangeable Oligonucleotide

Klarinda de Zwaan¹, Ran Huo¹, Myron N F Hensgens²

¹Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, 2629 HZ Delft, The Netherlands.

|March 27, 2025

Summary

This summary is machine-generated.

This study introduces a faster bioimaging workflow combining super-resolution optical fluctuation imaging (SOFI) and DNA-PAINT microscopy. This approach accelerates cellular structure imaging by 30-300x, enhancing throughput for biomolecular research.

Keywords:

DNA-PAINT blinking kinetics fluorescence fluctuation imaging high-throughput microscopy single-molecule localization microscopy super-resolution imaging

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Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Super-resolution microscopy offers molecular-level visualization but often suffers from low throughput.
Single-molecule localization microscopy (SMLM) techniques are accessible but limited in speed for large-scale screening.
Current limitations hinder the application of advanced microscopy in high-throughput biomolecular research.

Purpose of the Study:

To develop a workflow for efficient data collection in super-resolution microscopy.
To enhance the throughput of DNA-PAINT microscopy for cellular imaging applications.
To enable flexible adaptation of imaging resolution and acquisition time based on experimental needs.

Main Methods:

Exploited DNA oligo hybridization kinetics with DNA-PAINT probes for tailored fluorescent blinking.
Integrated fast fluctuation-based imaging (SOFI) with single-molecule localization microscopy (DNA-PAINT).
Developed a hybrid workflow starting with large-area scanning followed by targeted SMLM.

Main Results:

Achieved 30-300-fold faster imaging of cellular structures compared to conventional DNA-PAINT.
Demonstrated high-throughput SOFI imaging of large areas (0.65 mm × 0.52 mm) in 4 minutes.
Showcased flexible switching between high-throughput SOFI and high-resolution DNA-PAINT.

Conclusions:

Combining DNA-PAINT and SOFI offers a versatile approach for adaptable bioimaging resolution and speed.
The proposed workflow significantly improves throughput for SMLM techniques.
This method holds promise for applications in multiplexing and 3D super-resolution imaging.