Probing the dynamical properties of proteins at different spatial and temporal scales within functionnal living tissues – soLIVE

Cells can be considered as reactors in which the continuous biochemical interactions between proteins represent the network of interaction forming the physical and chemical basis of cellular behaviours. On one side, the recent advent in super-resolution microscopy has revolutionized our understanding of these cellular processes enabling the direct observation of proteins organization and dynamics at the molecular scale. On the other side, the development of Selective Plane Illumination Microscopy (SPIM) has offered the possibility to image thick live sample in 3D over prolonged duration. However, due to their respective illumination scheme, super-resolution microscopy is constrained in term of penetration depth, whereas the spatial resolution achievable by SPIM is limited. As a consequence, there is still no straightforward solution allowing 3D imaging of fast and slow proteins dynamics in a living tissue, at the single cell level with super-resolution capability.

In the current proposal, we aim at developing a turnkey 3D nanoscope with sub-cellular sectioning capabilities in order to monitor the organization and dynamics of molecular complexes at different time scales. To achieve this ambitious goal, we propose to combine on a unique instrumental setup two nanoscopy techniques, single molecule localization microscopy (SMLM) and structured illumination microscopy (SIM), with a single-objective SPIM architecture (soSPIM), recently developed in our group.

The soSPIM technique allows both fluorescence excitation and detection through a single high numerical aperture objective. It is based on the unique patented combination of micro-fabricated devices displaying 45° mirrors with a specific beam steering unit. While single molecule imaging at up to 30 µm above the coverslips has been demonstrated to be feasible, monitoring the dynamics of proteins at high spatial and temporal resolutions within complex tissues remains a challenging task. Such a new capability represents an important breakthrough and a step toward a better understanding of many cellular processes within complex tissues, where no satisfying solutions are yet available on the market. However, it involves the upgrade of the actual system through the implementation of excitation beam shaping approaches for extended field of view with reduced light sheet thickness and the development of SIM imaging in the soSPIM configuration.

In this project, we will take advantage of the simple and versatile optical architecture of the soSPIM, in order to develop a new generation of multimodal nanoscope, offering unique capabilities for monitoring protein organization and dynamics at different time scales, within complex biological samples. We will use our local expertise in microscopy, bioengineering, computer science and microfabrication, and work in close collaboration with academic and industrial partners that are already involved in the development and maturation of the soSPIM technique for a future commercialization.
In the framework of this proposal, we plan applying this innovative nanoscope for studying the role of adhesion sites during drosophila embryo development. This work will be performed in collaboration with the teams of G. Giannone at the IINS and N. Brown at the Gurdon Institute, both worldwide experts in the field. We expect deciphering the series of fast events during adhesion sites formation and their long term evolution during the different embryo development stages.

On a more general ground, this project will offer the opportunity for a young CNRS scientist recruited in 2015, to get some autonomy, and to enlarge the panel of techniques developed in the team, which are extensively used in the IINS and abroad to address specific and cutting edge questions in the fields of neuroscience and cell biology.