Development of photoreguated fluxional fluorophores for single-molecule imaging in living cells

Single molecule localization microscopy (SMLM) can be used to image live cells to reconstruct images and movies with nanometric resolution and gain information about cellular structures and dynamics. To detect single molecules, SMLM requires that the emitting and non-emitting states of a fluorophores are optically controlled using light. To fulfill this, high intensity laser irradiations are applied to the sample causing substantial damage to live cells (phototoxicity) and to the fluorescent dyes (photobleaching).

Prompted by this limitation, we developed a photoactivatable xanthene-based probe able to circumvent photobleaching. We designed the molecule to undergo thermal equilibrium between a non-emissive and emissive Z form. Activation was performed in a controlled manner using short pulses of 405 nm light that induced photoisomerization from a locked E to a fluxional Z form (Figure 1,A). SMLM experiments in live-cells showed that the number of emitting molecules could be detected indefinitely (561 nm channel) when sequential activation pulses were introduced before depleting the fluorescent population (~10 min). Because of the proton-transfer dependent equilibrium, the photoregulated fluxional fluorophore (PFF) 1 selectively stained acidic vesicles (e.g. lysosomes) found in mammalian cells (Figure 1,B). These vesicles were imaged with excellent spatiotemporal resolution in two and three dimensions (Figure 1,C).

The PFF system has great potential to visualize nanometric dynamics of live cells. Currently, we are working on expanding the scaffold to afford multiple colors and to develop pH independent, targetable PFF derivatives.

Figure 1. Overcoming photobleaching with photoregulated fluxional fluorophors. A. Mechanism of activation and chemical structure of PFF 1. Short-pulse irradiation of 405 nm light controls isomerization from the E to the Z form. Single molecules are detected by reading out fluorescence emission in the 561 nm imaging channel. B. Plot of the number of emitter molecules per frame in time detected over 30 min in a SMLM experiment performed with live HeLa cells treated with PFF 1 (500 nM). C. Time-lapse SMLM experiment from panel B shows stained lysosomes in live HeLa cells after: (top) a single photoactivation pulse (405 nm, 2.6 Wcm-2, 20 ms) and (bottom) sequential photoactivation pulses (405 nm, 2.6 Wcm-2, 20 ms) every 10 min. Brightfield images show HeLa cells prior to imaging. Region of interest (ROI) highlighted in green depicts the region in acquisition intervals of t = 0, 15 and 30 min (right).  The respective sub-diffraction limited image is shown at the bottom left corner (t = 0 min). Scale bars = 10 µm (brightfield), 500 nm (ROI).

Halabi, E. A.; Pinotsi, D.; Rivera-Fuentes, P. Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching. Nat. Commun. 2019, 10, 1232.

Development of targeted, photoactivatable phosphine-releasing probes for the induction of intracellular reductive stress

Reductive stress can be defined as a disruption of the redox homeostasis towards a more reducing environment and it has been associated with many pathologies, such as cancer, inflammation and neurodegenerative diseases.
For research purposes, reductive stress can be induced by pharmacological agents, such as dithiothrietol (DTT), but these lack selectivity and spatial resolution.
To overcome these shortcomings, we developed a photoactivatable probe that releases a trialkylphosphine as the reducing agent. Our probes can be activated with single-cell precision and they induce formation of protein aggregates, which are known to be a sign of reductive stress.
Current research focuses on improving the thermal stability of the phosphonium probes so they could be further modified for subcellular targeting.

A.Tirla, P. Rivera-Fuentes, Angew. Chem. Int. Ed.201655, 14709–14712.
Development of a Photoactivatable Phosphine Probe for Induction of Intracellular Reductive Stress with Single-Cell Precision.

Small-molecule, photoactivatable probes for super-resolution imaging of enzymatic activity in live cells

In this project, we explore the photochemical properties and applications of a double-activatable fluorophore 1. Upon enzymatic deacetylation and subsequent irradiation with 405 nm laser, 1 yields a major fluorescent photoproduct 2. In the absence of the preceding enzymatic deacetylation, however, mostly non-fluorescent compound 3 is obtained. A bright signal can thus only be attained after photoactivation of 1 in the presence of intracellular carboxylesterases. Moreover, under constant light irradiation, we expect fast photoconversion of 1 immediately after deacetylation. This mechanism is useful for in situ mapping of enzymatic reactions and tracking of carboxylesterases with stochastic optical reconstruction microscopy (STORM).


This principle was further extended to detect nitroreductase (NTR) activity. Nitroreductases are a class of enzymes that reduce aromatic nitro groups into their corresponding anilines and play a role in pro-drug activation and detoxification. Even though the presence of nitroreductase activity in solid tumors under hypoxic conditions is well studied, their role in healthy mammalian cells remains unclear. Using our probe, we could image nitroreductase activity in mammalian cells on a super-resolution scale and were able to show that this activity is predominantly present in microdomains within mitochondria. We are currently working on further identification of the enzymes displaying nitroreductase activity. 

E. A. Halabi*, Z. Thiel*, N. Trapp, D. Pinotsi, P. Rivera-Fuentes,J. Am. Chem. Soc.2017139, 13200–13207. 
A Photoactivatable Probe for Super-Resolution Imaging of Enzymatic Activity in Live Cells. (*These authors contributed equally)

Z. Thiel, P. Rivera-Fuentes, Angew. Chem. Int. Ed.2019, 53, 11474–11478
Single‐Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo‐Crosslinking Fluorescent Sensor