Programmable Fluorogenic Probes for Super-Resolution Imaging – PFPImaging

To improve temporal resolution, it is essential to reduce background noise. In this context, organic dyes have caught our attention as fluorescent reporters. Among organic fluorophores, we have particularly focused on those that «activate« their fluorescence intensity, also known as fluorogenic dyes, in response to interactions with their biomolecular targets. These dyes must produce a target-specific emission signal and ensure noise-free imaging. To maximize spatiotemporal resolution, these emissive dyes must also exhibit high brightness, photostability, and selectivity. Organic probes meeting all these conditions are rare. To address this challenge, we have explored 2 approaches. The first approach involves introducing environmentally sensitive fluorogenic probes. Thus, the imager strand, by binding to the complementary anchor strand, should lead to an environment where the fluorophore is less mobile and less hydrated. Among fluorophores sensitive to hydration and/or medium viscosity, different families of fluorophores have caught our attention. The second approach is based on a new concept we introduced, intermolecular DRET (DRET: Dark Resonance Energy Transfer). It involves synthesizing highly quenched probes and then hybridizing them onto an anchor strand labeled with a bright acceptor so that the quenched donor transfers its energy by resonance to the acceptor, thus enabling its detection.

Through click chemistry, we have developed a robust synthesis protocol for efficiently performing the covalent coupling of different fluorophores to imager strands. The nature of the coupling link and its specific position on the strand also played a crucial role in our selection process. In-depth characterization of absorption and emission properties highlighted two particularly interesting candidates for the fluorogenic approach and DRET. Our studies also allowed us to identify sequences producing the most significant signal enhancements. The most significant signal amplification was observed using DRET. This system was preferentially exploited for single molecule and super-resolution microscopy studies. We have developed a new imaging modality, which has been implemented on the PIQ-QUEST imaging platform, accredited by IBISA and part of the Alsace node of France BioImaging. It is thus open to a broad user community. Over the period, the majority of the initial objectives were achieved. Only the study of an environmentally sensitive fluorogenic probe at the single molecule level and in imaging was not carried out. However, this project has enabled us to establish a versatile and flexible synthesis protocol, allowing us to rapidly screen fluorescent reporters. This part of the project can therefore be pursued more efficiently. The most striking result of our work was achieved by introducing the super-resolution imaging modality in DRET. Compared to the 30 minutes required for the conventional system, it allows acquiring super-resolved images of cellular microtubules in just 30 seconds. Recently, other teams have published alternative solutions to DRET. Our system compares favorably with both modalities. However, a disadvantage of our system compared to these is that it does not allow image collection over such a long period due to acceptor photobleaching. Improvement avenues are conceivable to overcome this drawback and make DRET more efficient.

In conclusion, this project supported by the ANR has enabled us to design original fluorescent probes for photon microscopy and introduce a new microscopy modality. It paves the way for faster and more efficient applications in high-resolution cellular imaging. These promising results broaden the possibilities of research in cell biology and open the door to new advances in the study of molecular structures. This work has also paved the way for new collaborations with other teams aiming to exploit DRET for new applications.

Altogether, this work has resulted in 3 published articles and 3 articles submitted for publication or in preparation. The published articles focus on the development and characterization of the new fluorescence probes. One submitted article describes the results obtained in super-resolution imaging. These works have been presented orally 7 times at conferences or thematic workshops, including 3 at international venues, and as posters 4 times. They have also provided the basis for two theses, one in chemistry and one in biophysics.

The PFPImaging project describes the milestones and the collaborations needed for the synthesis of fluorescent fluorogenic probes for a better super-resolution imaging of cellular targets. The state of the arts describes the existent methodologies and their insufficiencies. PFPImaging answers to the described bottlenecks when following specific research blueprints. The new blinking probes are specific, more bright and exquisitely tuned and adaptable for the super-resolution imaging of nucleic acid and protein targets. The collaborations are describing the expertise of each partner: e.g. fluorescent nucleic acid chemistry for the project leader and molecular photo-physical detection and imaging in vitro and in cellulo. We likewise pursue the aim to considerably improve the visualization of cellular tubulin to confirm our new tool’s performances. In the future, these new tools the method is generalizable to the observation of proteins and nucleic acids of cytoplasm, nucleus and other cellular compartments.