Precision targeted ruthenium(II) luminophores; highly effective probes for cell imaging by stimulated emission depletion (STED) microscopy

Fluorescence microscopy has undergone a dramatic evolution over the past two decades with development of super-resolution far-field microscopy methods that break the light diffraction limited resolution of conventional microscopy, offering unprecedented opportunity to interrogate cellular processes at the nanoscale. However, these methods make special demands of the luminescent agents used for contrast and development of probes suited to super-resolution fluorescent methods is still relatively in its infancy. In spite of their many photophysical advantages, metal complex luminophores have not yet been considered as probes in this regard, where to date, only organic fluorophores have been applied. Here, we report the first examples of metal complex luminophores applied as probes for use in stimulated emission depletion (STED) microscopy. Exemplified with endoplasmic reticulum and nuclear targeting complexes we demonstrate that luminescent Ru(II) polypyridyl complexes can, through signal peptide targeting, be precisely and selectively delivered to key cell organelles without the need for membrane permeabilization, to give high quality STED images of these organelles. Detailed features of the tubular ER structure are revealed and in the case of the nuclear targeting probe we exploit the molecular light switch properties of a dipyrido[3,2-a:2',3'-c] phenazine containing complex which emits only on DNA/RNA binding to give outstanding STED contrast and resolution of the chromosomes within the nucleus. Comparing performance with a member of the AlexaFluor family commonly recommended for STED, we find that the performance of the ruthenium complexes is superior across both CW and gated STED microscopy methods in terms of image resolution and photostability. The large Stokes shifts of the Ru probes permit excellent matching of the stimulating depletion laser with their emission whilst avoiding anti-Stokes excitation. Their long lifetimes make them particularly amenable to gated STED, giving a much wider window for gating than traditional probes. Our findings indicate that ruthenium polypyridyl peptide targeted probes are a powerful new partner to STED microscopy, opening up new approaches to probe design for STED microscopy.