Jon Grimm: "To Azetidine and Beyond: Ten Years of Janelia Fluor Evolution."

Rhodamine dyes were first discovered nearly 150 years ago, yet they remain one of the premier scaffolds for fluorescent probes due to their excellent spectral properties, tunable structures, and bioavailability. Several landmark campaigns to improve dye performance afforded novel, popular classes of rhodamines with high brightness and solubility (e.g., Alexa Fluor), but these highly polar, cell-impermeant dyes forfeit the live-cell compatibility of “classic” rhodamines. At the same time, the rise of advanced fluorescence imaging techniques such as single-particle tracking (SPT) and specific, enzyme-based labeling strategies (e.g., self-labeling tag proteins) has driven the need for new dyes that retain cell permeability but improve upon the suboptimal brightness and photostability of the small set of classic, neutral rhodamines.
In 2014, we reported a simple structural modification that improves the spectral properties of rhodamines while preserving cell permeability: the replacement of N,N-dialkylamino groups with 4-membered azetidine rings. This effect is generalizable across many fluorophore scaffolds, yielding a diverse palette of azetidinyl “Janelia Fluor” (JF) dyes. Since then, we have refined this strategy to further tune the spectra, brightness, and photostability of rhodamines across the visible range. Substitution on the azetidine ring and replacement of the bridging oxygen atom with other heteroatoms allows for precise, rational tuning of color and chemical properties. Incorporation of deuterium into the dialkylamino auxochromic groups greatly enhances photostabililty. Fluorination of the bottom phenyl ring strongly shifts the lactone-zwitterion equilibrium of rhodamines to the fluorescent (zwitterionic) form while simultaneously enabling late-stage derivatization via novel, facile chemistry. Now, we report a significant expansion of these approaches to encompass a larger suite of cell-permeable rhodamine scaffolds beyond the traditional JF azetidinyl rhodamines, from green rhodamine 110 derivatives to live-cell compatible ATTO 647N analogs and near-infrared Si-rhodamines beyond 700 nm.
In this seminar, I will summarize the evolution of the Janelia Fluor dyes from the prototypical azetidinyl rhodamines through our most recent work expanding the fluorination strategy and other structural modifications to the wider array of rhodamine structures. I will also highlight the performance and advantages of these molecular tools in advanced biological imaging techniques, from SPT to super resolution and fluorescence lifetime imaging microscopy.