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.
