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Mechanically-Interlocked Ring-on-Thread Molecules for Super-Resolution Imaging

Periodic Reporting for period 1 - Ring-STORM (Mechanically-Interlocked Ring-on-Thread Molecules for Super-Resolution Imaging)

Reporting period: 2018-10-15 to 2020-10-14

Ring-STORM focuses on developing supramolecular systems for advancing the field of fluorescence imaging. For this, Ring-STORM integrates state-of-the-art concepts from supramolecular chemistry and photochemistry to develop functional molecules with tailored properties for microscopy, focusing on specific properties, which are relevant for potential biological applications.

Super-resolution / localization microscopy methods are usually performed using blinking fluorophores, either engineered photoactivable fluorescent proteins (PALM) or dyes immersed in blinking buffers, which are not routinely compatible with live cells (STORM). In live cells, state-of-the-art methods are mostly limited to studies of easily accessible membrane molecules by specific extracellular ligands imaged upon binding (Universal- or DNA-PAINT). Our Ring-STORM methodology, affording autonomous blinking molecular machines, will overcome major inherent limitations of current super-resolution imaging methods offering a powerful tool for biology and medicine. This makes Ring-STORM a possible candidate for developing new diagnosis tools based on optical imaging, benefitting society.

The novel “blinking” fluorescent molecular systems have an overall objective of offering a new localization microscopy method ("Ring-STORM") with potential uses in live cell imaging, without the need for overexpressing genetically encoded fluorescent proteins.
Ring-STORM is based on the development of nanometric rotaxane structures with the main objective of instilling tailored “blinking” in fluorescent organic molecules. In this goal, this three interconnected areas of expertise have been combined:

1. Molecular design and synthesis
Initially, the experience of Dr. Martí in organic synthesis and computational modelling has been key to design and synthesize functional molecular machines incorporating the required structural elements to achieve blinking properties. For this, Dr. Martí designed and synthesized different generations of molecular machines. The synthesis involved different molecular fragments such as water-soluble stoppers, speed-bumps to regulate the macrocycle shuttling speed, ultra-stable fluorophores and other elements to complete the molecular machine structure.

2. Photochemistry
After successfully completing the synthesis of the prototype blinking molecules, Dr Martí measured the photochemical properties and received advanced training in the primary host at Institut des Sciences Moléculaires (CNRS/Université de Bordeaux). Dr Martí performed studies using steady state fluorescence and time resolved fluorescence to determine the fluorescence intensity and lifetimes. Steady state fluorescence measurements allowed determining the OFF/ON ratios as the ratio of the fluorescence intensity of the molecular machine and a reference. The molecular machines with best performance were selected for single-molecule microscopy studies.

3. Single-molecule microscopy
The most performant molecular machines in the photochemistry experiments were tested in single-molecule super-resolution microscopy. Dr Martí received advanced training in the secondary host at Laboratoire Photonique Numérique et Nanosciences - Institut d'Optique Graduate School (CNRS/Université de Bordeaux) and developed a protocol for measuring molecular machine performance in super-resolution microscopy. The super-resolution microscopy experiments allowed determining the position of single molecules and also measuring the intensity of the emitted and single-molecule time-traces. Statistical analysis of the single-molecule time-traces allowed obtaining kinetic parameters and the most performant systems had a duty cycle of <2% in the absence of any blinking buffer. Therefore, the developed Ring-STORM molecular machines are ideal dyes for super-resolution microscopy with a high potential for in live cell imaging.

Ring-STORM results have ben disseminated through formal academic publications and communications in international conferences and seminars. During the course of the MSCA-IF action, different public engagement activities have been performed. For this, Dr. Martí participated in different outreach activities for disseminating his research work targeting a broad audience. These activities include the European researcher’s night 2019 in Bordeaux, the "Fete de la Science 2019" at Institut de Sciences Moleculaires in Bordeaux. These activities were complemented with an interview with the Society for the Improvement of Science (SACSIS) targeting a broad audience. In this interview Dr. Martí explained Ring-STORM project ambitions and progress and also the overall experience of participating in a MSCA-IF action.
Overall, the work developed in the three interconnected areas of expertise allowed developing state-of-the-art molecular machines to achieve Ring-STORM project objectives.
Through Ring-STORM, Dr. Martí developed unprecedented autonomous novel photoactive nanoscopic molecular machines that “blink” for implementation in fluorescence microscopy imaging with a resolution higher than the light scattering limit. Its use in super-resolution imaging represents a major innovation in imaging, both conceptually and practically, overcoming many inherent constraints associated with current techniques.

By the end of the project it is expected to demonstrate that it is possible to achieve a resolution <50 nm with the developed blinking molecular machines. In the near future it is expected to use the foundations and know-how developed in Ring-STORM project to develop tailored systems for specific biological applications and this could have a significant socio-economic impact. The new localization microscopy method ("Ring-STORM") is adapted for living cells without the need for overexpressing genetically encoded fluorescent proteins.

Ring-STORM also had a positive impact on the fellow career. The added value of training obtained by the project gave independence and professional maturity by the acquisition advanced experimental abilities, public engagement, knowledge sharing, and communication skills. Dr. Martí is one of the few experts in the EU in artificial nano-machine synthesis with extensive photochemistry training, placing him in a unique position to secure a professional research leadership position after the fellowship and successfully develop new research areas.
Ring_STORM single-molecule detection and the associated blinking time-trace.