Super-Photostability and Super-Brightness of EC5 Dyes for Super-Resolution Microscopy in the Deep Near-Infrared Spectral Region

The confocal and super-resolution imaging has become routine for the visible spectral region, but not for the deep near-infrared (NIR) region (800-1000 nm) due to the lack of bright and ultraphotostable fluorophores. We devised a premium fluorochromic scaffold (EC5) spectrally active in this region and further optimized it for super-resolution imaging by rational and systematic molecular engineering over the push-pull headgroups. EC5j maximally absorbs/emits at 835/873 nm and offers a superior brightness of 38070 cm^-1 M^-1 and superior photostability. Using two home-built microscopes with deep-NIR capability, i.e., confocal and structured-illumination microscopes, we demonstrated that super-resolution 2D and 3D-microscopy is now practical with EC5j. In particular, EC5j-adsorbed polystyrene beads furnished a notable spatial resolution of 175 nm with structure-illumination microscopy (SIM) imaging, as compared to the theoretical diffraction limit of 336 nm. Proof-of-concept applications included live-cell confocal imaging of mitophagy, SIM imaging of mitochondria, and deconvolution-based confocal sectioning of cell structures and brain vasculature. The revolutionary expansion of the imaging spectral window brings the field new tools and new insights.