Periodic Reporting for period 1 - TiFuN (Tiny Functional Au Nanorods: Novel NIR-Photothermal Nanoprobes for Single-Molecule Tracking at Confined Cellular Environment) Reporting period: 2015-04-01 to 2017-03-31 Summary of the context and overall objectives of the project Over the last decade, single-molecule optical microscopy has become the gold-standard approach to study the organization and dynamics of bio-molecules at the nanometer scale, and to decipher complex molecular processes in cellular environments. Developments in single molecule imaging involve improvements on two complementary aspects: the microscopy techniques allowing probe detection with always better spatial and temporal resolutions, and the probe synthesis allowing better biocompatibility, brightness (for precise detection), optical stability (for longer detection), specificity, monovalency together with a size as small as possible in order not to perturb the molecular function. Because of its high sensitivity, the most frequent single molecule techniques in biological environments are based on fluorescence. They benefit from a large toolbox of labeling strategies with fluorescent proteins, organic dyes or quantum dots. However, current fluorescence-based microscopies are however, limited by bleaching and blinking or by the probe size.This project aimed at developing an alternative approach to fluorescence for single biomolecule detection and tracking. It was based on the development of a photothermal imaging setup working at the biological optical window, and on the synthesis, functionalization and conjugation of tiny gold nanorods for biolabeling. Because they present strong optical absorption tunable from the red to the near IR, gold nanorods could be detected with high signal-to-noise and signal-to-background ratios at the single particle levels and in biological samples.We anticipated that small bio-conjugated nanorods will constitute the next generation photothermal probe to study complex molecular dynamics in biological systems owing to their small size, tunable NIR-absorption, absolute photostability, and chemical suitability for surface functionalization and bioconjugation.Ultimately the project aimed at opening a new way to study dynamics of biomolecules in confined cellular regions such as neurotransmitter receptors in synapses or integrin proteins in adhesion sites. This helps to reveal the molecular mechanisms of synaptic transmission, which are at the basis of synaptic plasticity and their deregulations are involved in pathologies including cognitive disorder. It also allows to study the nanoscale organization and dynamics of cell adhesion sites and actin networks which control critical cellular functions. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Overall aim of this project was to develop tiny nanorod probes for the photothermal optical imaging. Even though different methods are known for the synthesis of nanorods, development of tiny rods (T-rods; length/diameter < 10/5 nm) is bottleneck in nano-chemistry. To accomplish this step, we have used a pH-mediated seedless protocol. To improve nanorods monodispersity with tunable longitudinal plasmon resonance, we performed length sorting by density gradient ultracentrifugation protocol. Different fractions resulted in density gradient ultracentrifugation shows strong optical absorption tunable from the red to the near IR. We have demonstrated the use of these probes with PhI. Interestingly, we have seen better signal to background ratios from nanorods imaged in cells upon 640 nm as compared to 532 nm excitation. These nanoprobes show great promises for tackling biological applications described in the project. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In this project we developed a novel bioimaging paradigm based on near-infrared photothermal microscopy and tiny plasmonic gold probes.This opens a new way to study dynamics of biomolecules in confined cellular regions and decipher complex molecular mechanisms such as those occurring with neurotransmitter receptors in synapses or integrin proteins in adhesion sites. This will help to reveal the molecular mechanisms of synaptic transmission, which are at the basis of synaptic plasticity and their deregulations are involved in pathologies including cognitive disorder. It will also allow to study the nanoscale organization and dynamics of cell adhesion sites and actin networks which control critical cellular functions responsible of cancer cell proliferation. Shows microscopic image of new probe and photothermal image of cells at two different excitations
