resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Area of Science:

• Biophysics
• Microscopy
• Nanotechnology

Background:

• Localization microscopy techniques like PAINT offer high precision but face limitations.
• High probe concentrations in PAINT lead to background fluorescence, hindering volumetric imaging.
• Existing methods struggle with achieving high localization rates without compromising image contrast.

Purpose of the Study:

• To develop an advanced PAINT technique with improved probe accumulation and photophysics control.
• To overcome the limitations of conventional PAINT, particularly in high probe concentration scenarios.
• To enable high-resolution imaging of biological structures, including entire cell surfaces and membrane proteins.

Main Methods:

• Introduced reservoir-PAINT (resPAINT), integrating PAINT with active control of probe photophysics.
• Developed an activatable probe 'reservoir' that accumulates on target molecules.
• Combined resPAINT with large depth-of-field microscopy for volumetric super-resolution imaging.

Main Results:

• Achieved a 50-fold increase in localization rate compared to conventional PAINT.
• Maintained high image contrast despite increased probe accumulation.
• Demonstrated super-resolution imaging of entire cell surfaces using resPAINT.
• Successfully imaged membrane proteins using resPAINT with a Fab fragment.

Conclusions:

• resPAINT significantly enhances the performance of localization microscopy.
• The technique expands the applicability of PAINT for imaging diverse biological interactions and structures.
• resPAINT offers a powerful tool for high-resolution, high-speed imaging in biological research.