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

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• DNA-PAINT offers high multiplexing and molecular counting capabilities for nanoscale imaging.
• Challenges in target accessibility and variable imaging performance limit DNA-PAINT's broad application.
• Robust single-protein imaging and accurate molecular counting require optimized methodologies.

Purpose of the Study:

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

Main Methods:

• Integration of Total Internal Reflection Fluorescence (TIRF) microscopy with physical sectioning, specifically Tokuyasu cryosectioning.
• Development of tomographic & kinetically enhanced DNA-PAINT (tkPAINT).
• Optimization of imager binding and cellular integrity during cryosectioning and imaging.

Main Results:

• Achieved 3 nm localization precision across various sample types.
• Demonstrated enhanced imager binding and improved cellular integrity.
• Successfully quantified in situ RNA Polymerase II abundance within the nucleus of HeLa cells and mouse tissues.
• Showcased multiplexing, multimodal targeting (proteins and nucleic acids), and 3D imaging capabilities.

Conclusions:

• tkPAINT significantly enhances DNA-PAINT's utility for robust single-protein imaging and molecular counting.
• The method overcomes limitations in target accessibility and imaging performance.
• tkPAINT is a versatile tool for investigating biomolecular organization and interactions in cells and tissues.