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

Area of Science:

• Super-resolution microscopy
• Biophysics
• Nanotechnology

Background:

• Points for Accumulation in Nanoscale Topography (PAINT) enables extensive localization microscopy measurements.
• High probe concentrations in PAINT lead to background fluorescence, limiting volumetric imaging.
• Existing methods struggle with high probe densities and contrast in super-resolution imaging.

Purpose of the Study:

• To develop a novel super-resolution imaging technique that overcomes the limitations of conventional PAINT.
• To enhance localization rates and improve imaging contrast in challenging biological samples.
• To expand the applicability of PAINT for imaging cellular structures and interactions.

Main Methods:

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

Main Results:

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

Conclusions:

• resPAINT significantly advances localization microscopy by boosting probe accumulation and localization rates.
• The technique enables high-resolution imaging of large cellular structures and diverse biological interactions.
• resPAINT broadens the utility of PAINT for various super-resolution applications, including 3D imaging.