Laurent Cognet

Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors

The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.

NMDA receptor surface mobility depends on NR2A-2B subunits

The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.

Advances in live-cell single-particle tracking and dynamic super-resolution imaging

Resolving the movement of individual molecules in living cells by single particle tracking methods has allowed many molecular behaviors to be deciphered over the past three decades. These methods have increasingly benefited from advances in microscopy of single nano-objects such as fluorescent dye molecules, proteins or nanoparticles as well as tiny absorbing metal nanoparticles. In parallel to these efforts aiming at tracking ever smaller and more photostable nano-objects in living cells, the development of localization-based super-resolution imaging provided means to increase the number of single molecules tracked on a single cell. In this review we will present the most recent advances in the field.

“Hyper‐bright” Near‐Infrared Emitting Fluorescent Organic Nanoparticles for Single Particle Tracking

An efficacious strategy to obtain photostable Hyper-bright near-IR emitting Fluorescent Organic Nanoparticles (HIFONS) is reported. These HIFONs show excellent chemical and colloidal stability and retain their pristine nanostructure and brightness after incubation in cellular environments. They can be identified at the single particles level with a wide-field microscope, emerging as highly promising tools for applications in bionanotechnologies.

PROPERTIES OF MICROELECTROMAGNET MIRRORS AS REFLECTORS OF COLD RB ATOMS

Cryogenically cooled microelectromagnet mirrors were used to reflect a cloud of free-falling laser-cooled

Optical detection of cold atoms without spontaneous emission

{}^{85}\mathrm{Rb}

Single-molecule imaging in live cell using gold nanoparticles

atoms at normal incidence. The mirrors consisted of microfabricated current-carrying Au wires in a periodic serpentine pattern on a sapphire substrate. The fluorescence from the atomic cloud was imaged after it had bounced off a mirror. The transverse width of the cloud reached a local minimum at an optimal current corresponding to minimum mirror roughness. A distinct increase in roughness was found for mirror configurations with an even versus odd number of lines. These observations confirm theoretical predictions.

Imaging single metal-nanoparticles in cells by photothermal interference contrast

We have demonstrated nondestructive detection of cold atoms with a probe laser by a frequency-modulation spectroscopy technique. We were able to tune the probe laser and its sidebands far from atomic resonance to reduce the spontaneous emission to less than 0.2 photon per atom during detection.

Ultrashort Carbon Nanotubes That Fluoresce Brightly In The Near-Infrared

Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.

NIR-emitting molecular-based nanoparticles as new two-photon absorbing nanotools for single particle tracking

We have developed a photothermal method for far-field optical detection of nanometer-sized metal particles, combining high-frequency modulation and polarization interference contrast. We can image gold colloids down to 5 nm in diameter, with a signal-to-noise ratio higher than 10. This is a considerable improvement over commonly used optical methods based on resonance plasmon scattering which, for background reasons, are limited to particles of more than about 40 nm in diameter. We also show that in addition to its intrinsic sensitivity, our photothermal method is totally insensitive to non-absorbing scatterers as 10 nm nanoparticles can be imaged in cells.