Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin.

Nucleolin is a multifunctional protein involved in essential biological processes. To precisely localize it and unravel its different roles in cells, fluorescence imaging is a powerful tool, especially super-resolution techniques. Here, we developed polymer-aptamer probes, both small and bright, adapted to direct stochastic optical reconstruction microscopy (dSTORM). Well-defined fluorescent polymer chains bearing fluorophores (AlexaFluor647) and a reactive end group were prepared via RAFT polymerization. The reactive end-group was then used for the oriented conjugation with AS1411, a DNA aptamer that recognizes nucleolin with high affinity. Conjugation via strain-promoted alkyne/azide click chemistry (SPAAC) between dibenzylcyclooctyne-ended fluorescent polymer chains and 3'-azido-functionalized nucleic acids proved to be the most efficient approach. In vitro and in cellulo evaluations demonstrated that selective recognition for nucleolin was retained. Their brightness and small size make these polymer-aptamer probes an appealing alternative to immunofluorescence, especially for super-resolution (10-20 nm) nanoscopy. dSTORM imaging demonstrated the ability of our fluorescent polymer-aptamer probe to provide selective and super-resolved detection of cell surface nucleolin.

Innovative particle standards and long-lived imaging for 2D and 3D dSTORM.

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.