A Photoactivatable Probe for Super-Resolution Imaging of Enzymatic Activity in Live Cells

A dual-activatable,
fluorogenic probe was developed to sense esterase
activity with single-molecule resolution. Without enzymatic pre-activation,
the diazoindanone-based probe has an electron-poor core and, upon
irradiation, undergoes Wolff rearrangement to give a ring-expanded
xanthene core that is nonemissive. If the probe is pre-activated by
carboxylesterases, the tricyclic core becomes electron-rich, and the
photoinduced Wolff rearrangement produces a highly emissive rhodol
dye. Live-cell and solution studies confirmed the selectivity of the
probe and revealed that the photoactivated dye does not diffuse away
from the original location of activation because the intermediate
ketene forms a covalent bond with surrounding macromolecules. Single-molecule
localization microscopy was used to reconstruct a super-resolved image
of esterase activity. These single-molecule images of enzymatic activity
changed significantly upon treatment of the cells with inhibitors
of human carboxylesterase I and II, both in terms of total number
of signals and intracellular distribution. This proof-of-principle
study introduces a sensing mechanism for single-molecule detection
of enzymatic activity that could be applied to many other biologically
relevant targets.