Cryosectioning-enhanced super-resolution microscopy for single-protein imaging across cells and tissues

Area of Science:

• Molecular Biology
• Biophysics
• Microscopy

Background:

• DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) offers high multiplexing and molecular counting capabilities for nanoscale imaging.
• Challenges in variable imaging performance and target accessibility limit the broader application of DNA-PAINT.
• Robust single-protein imaging and molecular counting require optimized methods for sample preparation and imaging techniques.

Purpose of the Study:

• To enhance the performance of DNA-PAINT for robust single-protein imaging and molecular counting.
• To improve target accessibility and imaging consistency in diverse biological samples.
• To develop a versatile tool for exploring biomolecular organization and interactions in cells and tissues.

Main Methods:

• Integration of total internal reflection fluorescence microscopy with physical sectioning, specifically Tokuyasu cryosectioning.
• Development of tomographic and kinetically enhanced DNA-PAINT (tkPAINT).
• Application of tkPAINT for in situ molecular counting within the nucleus and multiplexed imaging.

Main Results:

• Achieved 3 nm localization precision across diverse samples using tkPAINT.
• Demonstrated enhanced imager binding and improved cellular integrity.
• Successfully quantified the in situ abundance of RNA Polymerase II in HeLa cells and mouse tissues.
• Showcased tkPAINT's capacity for multiplexing, multimodal targeting, and 3D imaging.

Conclusions:

• tkPAINT significantly enhances DNA-PAINT's capabilities for nanoscale imaging, protein quantification, and multiplexing.
• The method provides high precision and improved performance for studying biomolecular organization in various biological contexts.
• tkPAINT is a versatile tool for advancing research in molecular biology, biophysics, and advanced microscopy techniques.