Abstract

We found that hydroxymethyl rhodamine green (HMRG) strongly fluoresces, while mono-amidated HMRG derivatives are colorless and non-fluorescent due to the preferred spirocyclized structure in aqueous solution at pH 7.4. Based on these findings, we have established a rational design strategy for novel fluorogenic probes, and developed various aminopeptidase-sensitive probes which were applicable for living cell system, including gGlu-HMRG, a novel HMRG-based “activatable” fluorescence probe for γ-glutamyltranspeptidase (GGT). We could detect tiny tumors in vivo by spraying gGlu-HMRG onto tissue surfaces that are suspected of harboring tumors, creating high signal contrast between the tumor and the background within 1 min. We also succeeded to develop a first-in-class spontaneously blinking fluorophore for single-molecule localization microscopy (SLM) imaging to construct super-resolution images, by optimization of intramolecular nucleophile and rhodamine-based fluorophore. By taking advantage of its spontaneous blinking regardless of laser intensity, SLM of nuclear pore structures far above from coverslip under spinning-disk confocal microscopes and repetitive time-lapse super-resolution imaging of microtubules in live cells could be achieved. Thus, our precisely designed spontaneously blinking fluorophore has a potential to expand the application of SLM deep into the cells and to track the motion of certain structure in living cells.

© 2015 Optical Society of America