Dr Jean-Baptiste Sibarita and Professor Daniel Choquet, University of Bordeaux : "Pushing the limits of SMLM toward deeper, more colours and more throughput imaging" and "The interplay between synapse nanoscale organization and function"

Speakers: 

11:00 - 11:45am

Dr Jean-Baptiste Sibarita

The Institute for Interdisciplinary Neuroscience, University of Bordeaux

Title: "Pushing the limits of SMLM toward deeper, more colours and more throughput imaging"

Abstract: The recent advent of optical super-resolution techniques represents a new and fundamental step toward understanding biological mechanisms at the molecular level in single cells. Single molecule-based approaches offer the capabilities to count, locate and track the movement of bio-molecules in their cellular environment. However, conventional illumination schemes used with these approaches limit the super-resolution observation to the first micron above the coverslip, preventing imaging live dynamical processes in whole 3D cells or tissues. In addition, monitoring multiple fluorescent species is often achieved at the expense of a loss in spatial and temporal resolutions, conventional experimental setups being typically limited to two simultaneous wavelengths. Finally, despite its high spatial resolution and unique quantitative information, SMLM remains a low throughput technique incompatible with HCS standards.

We will present our projects aiming at overcoming some of these limits, pushing SMLM toward deeper, more colours and more throughput. We will first present our last progress combining single objective Selective Plane Microscopy (soSPIM), adaptive optics (AO) and SMLM to achieve super-resolution imaging of cellular structures up to few tens of micrometers away from the coverslip. We will then show how, by employing a dual-objective imaging configuration compatible with live cell imaging, we can achieve simultaneous 3D single particle tracking of multiple distinct proteins without compromising the spatio-temporal resolution. Finally, we will describe a fully automated quantitative single-molecule-based super-resolution methodology operating in 96-well plates and using HCS based analysis and data mining software in a single workflow.

11.45 - 12.30pm

Professor Daniel Choquet

The Institute for Interdisciplinary Neuroscience, University of Bordeaux

Title: "The interplay between synapse nanoscale organization and function"

Abstract: The spatio-temporal organization of neurotransmitter receptors in the postsynaptic membrane is a fundamental determinant of synaptic transmission and thus information processing by the brain. Ionotropic AMPA glutamate receptors (AMPAR) mediate fast excitatory synaptic transmission in the central nervous system. Using a combination of high resolution single molecule superresolution imaging techniques and video-microscopy, we have established that AMPARs are not stable in the synapse as thought initially, but undergo continuous entry and exit to and from the post-synaptic density through lateral diffusion and that AMPAR are highly concentrated inside synapses into a few clusters of around seventy nanometers. These results open the new possibility that glutamatergic synaptic transmission is controlled by the regulation at the nanometer scale of the position and composition of these highly concentrated nanodomains. We demonstrate that AMPAR conformation strongly impacts their mobility, and that AMPAR surface diffusion directly controls both short and long term synaptic plasticity.