Extremely bright, near-IR emitting spontaneously blinking fluorophores enable ratiometric multicolor nanoscopy in live cells

New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multi-color, live-cell, single-molecule localization microscopy (SMLM), especially for experiments that demand ultra-high-resolution live imaging. Here we report the design, synthesis, and biological evaluation of Yale676sb, a photostable, near-IR emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.