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

• Published in: Angewandte Chemie International Edition Vol. 61; no. 42; pp. e202206919 - n/a
• Main Authors: Sanders, Edward W., Carr, Alexander R., Bruggeman, Ezra, Körbel, Markus, Benaissa, Sarah I., Donat, Robert F., Santos, Ana M., McColl, James, O'Holleran, Kevin, Klenerman, David, Davis, Simon J., Lee, Steven F., Ponjavic, Aleks
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 17.10.2022; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Active control; Biophysics; DNA; Emissions control; Fluorescence; Imaging techniques; Imaging, Three-Dimensional; Localisation Microscopy; Localization; Membrane Proteins; Microscopy; Microscopy, Fluorescence - methods; PAINT; Reservoirs; Single Molecule Imaging - methods; Single-Molecule Imaging; Super-Resolution Microscopy; PAINT; Biophysics; Localisation Microscopy; Single-Molecule Imaging; Super-Resolution Microscopy
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202206919

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir‐PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe “reservoir” accumulates on target, enabling a 50‐fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth‐of‐field microscopy, we demonstrate super‐resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions. A new super‐resolution technique for localisation microscopy, which combines active control of probe photophysics with stochastic binding is reported. resPAINT yields an up to 50‐fold improvement in localisation rate vs. PAINT without compromising contrast and is fully compatible with large depth of field imaging techniques. This opens the door to larger scale 3D localisation microscopy as imaging that normally takes days can now be completed in hours.