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

Abstract

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.

A Simple Probe for Super‐Resolution Imaging of the Endoplasmic Reticulum in Living Cells

“…Upon irradiation, the diazoindanone would undergo Wolff rearrangement to give a brightly fluorescent rhodamine dye. [10,11] We studied the photophysical properties of ER-1 using absorption and fluorescence spectroscopy (Figure 1,a) in phosphate buffered saline (PBS, pH = 7.4) and measured an approximate 150-fold increase in fluorescence emission after 15 min of irradiation (405 nm, 2 mW cm À 2 , 10 min). The emission signal appeared to reach saturation after 15 min of irradiation under these same conditions (Figure 1,b).…”

“…We reported the use of diazoindanone rhodols as effective dual photoactivatable dyes that emit a fluorescent signal selectively in the presence of an active carboxylesterase. [11] This method enables the detection of clusters of active enzymes because the photoactivated dyes form a covalent bond with their surroundings, which limits their diffusion and facilitates SMLM.…”

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

“…To confirm the identity of the putative photoproduct, we prepared compound 3 ( Figure 1C), which we expected to be the major photoproduct. [8,15,16] Liquid chromatography coupled with mass spectrometry (LC-MS) confirmed that compound 3 is indeed the major photoproduct formed upon irradiation of 2 in water ( Figure S5). Thesame results were obtained when compound 2 was irradiated with 405 nm light, which is commonly used in fluorescence microscopy ( Figure S6).…”

“…Even though super-resolution microscopy techniques such as stochastic optical reconstruction microscopy (STORM) [6] and photoactivation localization microscopy (PALM) [7] are well established, there is al ack of fluorescent sensors that are specifically optimized for these techniques.W er ecently developed as ensing mechanism that is compatible with STORM. [8] We reported that photoactivatable diazoindanone-modified xanthene dyes bearing electron-withdrawing substituents mainly form nonfluorescent photoproducts upon irradiation. Photoactivation following enzymatic transformation of the substituent into an electron-donating group yields highly fluorescent photoproducts that display minimal diffusion.…”

“…Photoactivation following enzymatic transformation of the substituent into an electron-donating group yields highly fluorescent photoproducts that display minimal diffusion. [8] Herein, we apply this principle to develop aprobe for the detection of nitroreductase activity in mammalian cells. Nitroreductases (NTRs) are ac lass of enzymes that catalyze the reduction of nitroaromatic compounds to their corresponding anilines.…”