Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds. As a proof-of-principle, fluorophores containing a borinate (RF620) or silanediol (SiOH2R) functionality at the bridging position of the xanthene ring system are developed as endogenous H2O2 sensors. Both these fluorophores display far-red to near-infrared excitation and emission prior to reaction. Upon oxidation by H2O2 both sensors are chemically converted to tetramethylrhodamine, producing significant (≥66 nm) blue-shifts in excitation and emission maxima. This work provides a new concept for the development of ratiometric probes.
Electrical potential differences
across lipid bilayers play foundational
roles in cellular physiology. Plasma membrane voltage is the most
widely studied; however, the bilayers of organelles like mitochondria,
lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide
opportunities for ionic compartmentalization and the generation of
transmembrane potentials. Unlike plasma membranes, organellar bilayers,
cloistered within the cell, remain recalcitrant to traditional approaches
like patch-clamp electrophysiology. To address the challenge of monitoring
changes in organelle membrane potential, we describe the design, synthesis,
and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter)
for optically monitoring membrane potential changes in the ER of living
cells. We pair a tetrazine-quenched RhoVR for voltage sensing with
a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting
to the ER. Bright fluorescence is observed only at the coincidence
of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target
fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO
is voltage-sensitive and that the LUnAR RhoVR can be targeted to an
intact ER in living cells. Using the LUnAR RhoVR, we use two-color,
ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release
from internal stores. Finally, we use the LUnAR RhoVR to directly
visualize functional coupling between the plasma–ER membranes
in patch clamped cell lines, providing the first direct evidence of
the sign of the ER potential response to plasma membrane potential
changes. We envision that the LUnAR RhoVR, along with other existing
organelle-targeting TCO probes, could be applied widely for exploring
organelle physiology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.