Brahim Lounis

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.

Optical manipulation of single flux quanta

Magnetic field can penetrate into type II superconductors in the form of Abrikosov vortices, which are magnetic flux tubes surrounded by circulating supercurrents often trapped at defects referred to as pinning sites. Although the average properties of the vortex matter in superconductors can be tuned with magnetic fields, temperature or electric currents, handling of individual Abrikosov vortices remains challenging and has been demonstrated only with sophisticated scanning local probe microscopies. Here we introduce a far-field optical method based on local heating of the superconductor with a focused laser beam to realize a fast and precise manipulation of individual vortices, in the same way as with optical tweezers. This simple approach provides the perfect basis for sculpting the magnetic flux profile in superconducting devices like a vortex lens or a vortex cleaner, without resorting to static pinning or ratchet effects.

Single-molecule imaging in live cell using gold nanoparticles

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.

Imaging single metal-nanoparticles in cells by photothermal interference contrast

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.

Ultrashort Carbon Nanotubes That Fluoresce Brightly In The Near-Infrared

The intrinsic near-infrared photoluminescence observed in long single-walled carbon nanotubes is known to be quenched in ultrashort nanotubes due to their tiny size as compared to the exciton diffusion length in these materials (>100 nm). Here, we show that intense photoluminescence can be created in ultrashort nanotubes (∼40 nm length) upon incorporation of exciton-trapping sp3 defect sites. Using super-resolution photoluminescence imaging at <25 nm resolution, we directly show the preferential localization of excitons at the nanotube ends, which separate by less than 40 nm and behave as independent emitters. This unexpected observation opens the possibility to synthesize fluorescent ultrashort nanotubes-a goal that has been long thought impossible-for bioimaging applications, where bright near-infrared photoluminescence and small size are highly desirable, and for quantum information science, where high quality and well-controlled near-infrared single photon emitters are needed.

Cryogenic single nanocrystal spectroscopy: reading the spectral fingerprint of individual CdSe quantum dots

Spectroscopically resolved emission from single nanocrystals at cryogenic temperatures provides unique insight into photophysical processes that occur within these materials. At low temperatures the emission spectra collapse to narrow lines revealing a rich spectroscopic landscape and unexpected properties, completely hidden at the ensemble level. Since these techniques were first used, the technology of nanocrystal synthesis has matured significantly and new materials with outstanding photophysical stability have been reported. Here we review our recent work that shows how cryogenic spectroscopy of single nanocrystals probes the fundamental excitonic structure of the band edge, revealing spectral fingerprints that are highly sensitive to a range of photophysical properties as well as nanocrystal morphology. In particular, spectral and temporal signatures of biexciton and trion emission are revealed and their relevance to emerging technologies discussed. In addition, we show how high resolution excitation spectroscopy can provide information on external processes that ultimately limit the coherence of the nanocrystal band-edge states. Overall we demonstrate how cryogenic single nanocrystal spectroscopy can be used as a vital tool for understanding fundamental photophysics and guiding the synthesis of new nanocrystal materials.

Using Optical Lattice for STED Parallelization

Being a scanning microscopy, Stimulated Emission Depletion (STED) needs to be parallelized for fast wide-field imaging. Here, we achieve large parallelization of STED microscopy using well-designed Optical Lattice (OL) for depletion, together with a fast camera for detection. Depletion optical lattices with 100 intensity “zeros” are generated by four-beam interference. Scanning only a unit cell, as small as 290 nm by 290 nm, of the depletion OL is sufficient for STED imaging. The OL-STED microscopy acquires super-resolution images with 70 nm resolution and at the speed of 80 ms per image.

Photothermal detection and tracking of individual non-fluorescent nano-objects in live cells

To overcome the photobleaching problem inherent to fluorescence techniques we recently developed a new optical detection method for individual non-fluorescent nano-objects. It allows detecting the movement of individual membrane proteins labeled with 5 nm gold nanoparticles in living cells for arbitrary long times.