Bis(trifluoromethyl)carborhodamines: Highly Fluorogenic, Far-Red to Near-Infrared Dyes for Live Cell Fluorescence Microscopy, Activity-Based Sensing, and Single-Molecule Microscopy

groups render BF dyes fluorogenic, by shifting the position of the open-closed equilibrium of the colorless lactone and colored zwitterion form, resulting in up to a 30-fold improvement in fluorogenicity compared to silicon-bridged rhodamines. In this paper, we present the design and computational analysis of BF dyes; synthetic studies to access over a dozen new BF dyes through a unique, late-stage functionalization strategy; spectra characterization; and applications in advanced fluorescence microscopy including no-wash intracellular labeling, functional imaging with chemigenetic indicators, and single molecule tracking in living cells. Together, this report shows that bis(trifluoromethyl)carborhodamine dyes provide a complementary approach to achieving long-wavelength, fluorogenic dyes for live cell microscopy that do not rely on dimethyl silicon rhodamines.