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Updated: Aug 12, 2026
Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment
Published on: January 6, 2026
PAINTpro: Protein-Based PAINT in Living Cells Using Engineered Reversible Bilirubin-Binding Fluorescent Protein
Min Zhu1, Yukang Zhang2, Luhao Zhang1,3
1Department of Biomedical Engineering, State Key Laboratory of Extreme Photonics and Instrumentation, Key Laboratory of Biomedical Engineering of Ministry of Education, Zhejiang Key Laboratory of Intelligent Sensing Technology and Advanced Medical Instrument, Zhejiang University, Hangzhou, China.
We developed PAINTpro, a novel protein-based live-cell super-resolution microscopy technique. This method enables nanoscale visualization of dynamic cellular structures with high resolution and extended imaging times.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Single-molecule localization microscopy (SMLM) techniques like Point Accumulation for Imaging in Nanoscale Topography (PAINT) offer nanometer resolution for observing subcellular structures.
- Live-cell SMLM imaging is hindered by limitations in labeling strategies and challenges in imaging dynamic structures over extended durations.
Purpose of the Study:
- To introduce PAINTpro, a protein-based PAINT technique for live-cell SMLM imaging of dynamic nanoscale structures.
- To enhance PAINT imaging capabilities for extended live-cell observation.
Main Methods:
- Development of PAINTpro utilizing an engineered reversible bilirubin (BR)-binding protein, Fluorescent protein for Maximizing Imaging with Nano-resolution (FluoroMIN), derived from UnaG.
- Incorporation of mutations in FluoroMIN, guided by in silico molecular dynamics simulations, to accelerate BR exchange and improve fluorescence properties.
- Application of PAINTpro for live-cell labeling and super-resolution imaging of dynamic cellular structures, including microtubules.
Main Results:
- PAINTpro achieved live-cell super-resolution PAINT imaging with a resolution of approximately 50 nm.
- The technique enabled long-term imaging of dynamic structures, providing time-resolved super-resolution images of microtubules up to 60 nm resolution.
- FluoroMIN demonstrated reversible fluorescence, high single-molecule signal yield, and prolonged switching lifetime.
Conclusions:
- PAINTpro is a robust protein-based PAINT technique facilitating live-cell SMLM imaging of dynamic nanoscale structures.
- The engineered FluoroMIN protein enhances PAINT imaging performance, enabling high-resolution, long-term visualization of cellular dynamics.
- PAINTpro offers adaptability to existing protocols, presenting a valuable tool for super-resolution biological process visualization.
