Developing new methods for super-resolution imaging in live-cell

Versatile super-resolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. In this study, I developed in collaboration with the group of L. Cognet (LP2N, Institut d’Optique, Bordeaux, France) and D. Choquet (IINS) a new method to obtain super-resolved images and diffusion maps of membrane proteins on living cells (Giannone et al., Biophys J. 2010). This method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of membrane biomolecules. We show that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.

By combining dual-color uPAINT with single-molecule FRET it is now possible to study at the nanoscale ligand-activated receptor dimers in live cells (Winckler et al., Sci. Rep. 2013).The use of non-neuronal model cell system (fibroblasts) was necessary for the development of uPAINT. In neurons, uPAINT contributed to the demonstration that glutamate receptors in dendritic spines are organized into nano-domains (Nair et al., J Neuro 2013).

Dynamic super-resolution imaging of endogenous proteins on living cells at ultra-high density.
Giannone, G., Hosy, E., Levet, F., Constals, A., Schulze, K., Sobolevsky, A. I., Rosconi, M. P., Gouaux, E., Tampé, R., Choquet, D. and Cognet, L.
Biophys J. 2010. 99, 1303-1310.

High-Content Super-Resolution Imaging of Live Cell by uPAINT.
Giannone G., Hosy E., Sibarita J.B., Choquet D., and Cognet L.
Methods in Molecular Biology. 2012. 950, 95-110.

Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95.
Nair D., Hosy E., Petersen J., Constals A., Giannone G., Choquet D., Sibarita J.B.
J Neuroscience. 2013. 33, 13204-13224.

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells.
Winckler P., Lartigue L., Giannone G., De Giorgi F., Ichas F., Sibarita J.B., Lounis B. and Cognet L.
Sci.Rep. 2013. 3, 2387.

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Developing new nano-probes for super-resolution imaging and single-molecule tracking in live-cell

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. In this study we have developed in collaboration with the group of B. Lounis (LP2N, Institut d’Optique, Bordeaux, France) a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies, called GFP-nanobodies, that recognize widely used green fluorescent proteins (GFPs) with a very high affinity (Leduc et al., Nano Letters 2013). These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

A highly specific gold nanoprobe for live-cell single-molecule imaging.
Leduc C., Si S., Gautier J., Soto-Ribeiro M., Wehrle-Haller B., Gautreau A., Giannone G., Cognet L., Lounis B.A.
NanoLetters. 2013. 13, 1489–1494