On-target photoassembly of pyronin dyes for super-resolution microscopy

Controlled photoactivation is an auspicious and emerging approach in super-resolution microscopy, offering virtually zero background signal from the marker prior to activation. Pyronins are well-established fluorophores, but due to their inherent intercalating tendency towards nucleic acids, their use has been mostly avoided in super-resolution microscopy. Here we describe a new class of diaryl ether and diaryl silane molecules that upon photoactivation close into fluorescent (silicon-)pyronins and term them PULI (Pyronin Upon Light Irradiation). This concept exploits the outstanding photophysical properties of pyronins (bright, photostable, and optimal spectral features for standard microscopes), while overcoming their major drawback (intrinsic affinity of accumulating in the nucleus and around RNA) for the design of fluorescent markers for imaging applications. Furthermore, we also demonstrate that this approach is applicable to their Si-bridged analogues, extending this family of photoactivatable molecules to the far-red regime. The versatility of our approach was also highlighted by tagging diverse biological targets in cells and visualizing using advanced super-resolution microscopy techniques, such as PALM, STED, and MINFLUX.