Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensi
ons and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Forster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.
While addition of electrolyte to sodium dodecyl sulfate suspensions of single-wall carbon nanotubes has been demonstrated to result in significant brightening of the nanotube photoluminescence (PL), the brightening mechanism has remained unresolved.
Here, we probe this mechanism using time-resolved PL decay measurements. We find that PL decay times increase by a factor of 2 on addition of CsCl as the electrolyte. Such an increase directly parallels an observed near-doubling of PL intensity, indicating the brightening results primarily from changes in nonradiative decay rates associated with exciton diffusion to quenching sites. Our findings indicate that a reduced number of these sites results from electrolyte-induced reorientation of the surfactant surface structure that partially removes pockets of water from the tube surface where excitons can dissociate, and thus underscores the contribution of interfacial water in exciton recombination processes.
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. Due to these issues, long-term tracking of proteins in confined and crowded environments,
such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. 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.
Single-nanotube photometry was used to measure the product of absorption cross-section and fluorescence quantum yield for 12 (n,m) structural species of semiconducting SWNTs in aqueous SDBS suspension. These products ranged from 1.7 to 4.5 x 10(-19)
cm2/C atom, generally increasing with optical band gap as described by the energy gap law. The findings suggest fluorescent quantum yields of ~8% for the brightest, (10,2) species and introduce the empirical calibration factors needed to deduce quantitative (n,m) distributions from bulk fluorimetric intensities.
Single-molecule chemical reactions with individual single-walled carbon nanotubes were observed through near-infrared photoluminescence microscopy. The emission intensity within distinct submicrometer segments of single nanotubes changes in discrete
steps after exposure to acid, base, or diazonium reactants. The steps are uncorrelated in space and time, and reflect the quenching of mobile excitons at localized sites of reversible or irreversible chemical attack. Analysis of step amplitudes reveals an exciton diffusional range of about 90 nanometers, independent of nanotube structure. Each exciton visits approximately 104 atomic sites during its lifetime, providing highly efficient sensing of local chemical and physical perturbations.
The Photothermal Heterodyne Imaging method is used to study for the first time the absorption spectra of individual gold nanoparticles with diameters down to 5 nm. Intrinsic size effects wich result in a broadening of the Surface Plasmon resonance ar
e unambiguously observed. Dispersions in the peak energies and homogeneous widths of the single particle resonances are revealed. The experimental results are analysed within the frame of Mie theory.
We introduce a new, highly sensitive, and simple heterodyne optical method for imaging individual nonfluorescent nanoclusters and nanocrystals. A 2 order of magnitude improvement of the signal is achieved compared to previous methods. This allows for
the unprecedented detection of individual small absorptive objects such as metallic clusters (of 67 atoms) or nonluminescent semiconductor nanocrystals. The measured signals are in agreement with a calculation based on the scattering field theory from a photothermal-induced modulated index of refraction profile around the nanoparticle.
Current methods for producing single-walled carbon nanotubes (SWNTs) lead to heterogeneous samples containing mixtures of metallic and semiconducting species with a variety of lengths and defects. Optical detection at the single nanotube level should
thus offer the possibility to examine these heterogeneities provided that both SWNT species are equally well detected. Here, we used photothermal heterodyne detection to record absorption images and spectra of individual SWNTs. Because this photothermal method relies only on light absorption, it readily detects metallic nanotubes as well as the emissive semiconducting species. The first and second optical transitions in individual semicontucting nanotubes have been probed. Comparison between the emission and absorption spectra of the lowest-lying optical transition reveal mainly small Stokes shifts. Side bands in the near-infrared absorption spectra are observed and assigned to exciton-phonon bound states. No such sidebands are detected around the lowest transition of metallic nanotubes.
Photothermal heterodyne detection is used to record the first room-temperature absorption spectra of single CdSe/ZnS semiconductor nanocrystals. These spectra are recorded in the high cw excitation regime, and the observed bands are assigned to trans
itions involving biexciton and trion states. Comparison with the single nanocrystals photoluminescence spectra leads to the measurement of spectral Stokes shifts free from ensemble averaging.
