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.
