State Selective Pumping Reveals Spin-Relaxation Pathways in CdSe Quantum Dots

The band-edge exciton in elongated CdSe nanocrystals is composed of an upper and lower manifold associated with heavy and light holes in which the energy separation is sensitive to the nanocrystal shape. Using resonant photoluminescence excitation, we probe the upper heavy hole exciton manifold and find rapid relaxation to the lower light hole manifold on a 5 ps time scale. State selective excitation allows the preparation of single quantum states in this system. We used this to map the hole spin relaxation pathways between the fine structure sublevels, which have energy splittings incommensurate with either optical or acoustic phonon energies. This reveals a hitherto unexpected hole spin-relaxation channel in these materials

Metrological Investigation of the (6,5) Carbon Nanotube Absorption Cross Section

Using single-nanotube absorption microscopy, we measured the absorption cross section of (6,5) carbon nanotubes at their second-order optical transition. We obtained a value of 3.2 × 10^–17 cm²/C atom with a precision of 15% and an accuracy below 20%. This constitutes the first metrological investigation of the absorption cross section of chirality-identified nanotubes. Correlative absorption–luminescence microscopies performed on long nanotubes reveal a direct manifestation of exciton diffusion in the nanotube

FGF2-NP is mobile in the pericellular matrix of Rama 27 fibroblasts.

<p>FGF2-NP (22 pM) was incubated for 30 min with Rama 27 fibroblasts before washes and image acquisition by PHI. (A) Image of 10×10 µm part of a living cell. The <i>x</i>- and <i>y</i>-axes correspond to the relative position of this picture within a 100 µm×100 µm image that was acquired first (not shown). Static FGF2-NP molecules appear as bright spots (circle), while ones moving along the direction of the scan (<i>x</i>-axis) appear as short lines (rectangle), due to the scanning image acquisition mode. (B and C) Images acquired in the same 10×10 µm area of a fixed cell at two different time points (interval of 70 min). While some FGF2-NP molecules are static (circle) others have moved (rectangle).</p

Schematic representation of the HSPG in the pericellular matrix.

<p>In the crowded pericellular matrix (macromolecular concentration of ∼400 mg/mL), proteins and proteoglycans interact with each other. Only HSPG proteoglycans (in black and grey), the FGF2 (in green) and few other HS partners (in blue), are represented here for clarity. The HS-binding partners, which may be membrane-associated or “soluble” in the matrix, include growth factors, cytokines, chemokines, enzymes, matrix proteins, and numerous cell-surface receptors. Proteoglycan core proteins (black, inserted in the membrane) are shown with their HS chains (dark grey lines), which are between 40 to 160 nm long. Along these chains, dark grey rectangles represent strong binding sites for FGF2, and light grey rectangles represent weaker binding sites for FGF2. These binding sites form non-random networks of heterogeneously distributed binding sites within which the FGF2 moves by translocating from one site to another (a). A path of the FGF2 is shown by green arrows and some of its successive positions marked as a green circle. This motion of the FGF2 is independent of the motion of the protein core of the HSPG itself (b). Movement of the HS chains (c) to which the FGF2 is attached may also contribute to the motion of the FGF2 within the pericellular matrix. Note that the many endogenous binding partners of HS chains and HSPG core proteins may, in some conditions, severely restrict the diffusion of the protein core and of the HS chains (asterisks).</p

FGF-2 NP in the pericellular matrix are clustered.

<p>TEM of plasma membrane sheets reveals clustering and the heterogeneous spatial distribution of FGF2-NP at high resolution. Five hundred and fifty pM (A) or 2.8 nM (B) of FGF2-NP were added to living cells before washing, plasma membrane sheet preparation, and fixation. No labelling was observed when using 2.8 nM of control nanoparticles (non-specific binding control with NP-TrisNiNTA, not conjugated to FGF2) (C). (D) 2.8 nM FGF2-NP in the presence of 50 µg/mL heparin-derived dodecasaccharide (DP12). In this condition, FGF-2 binding to the HS of the pericellular matrix was abolished, but not the interaction with the FGFR. The little labelling that was observed corresponded to complexes of FGF2-NP with FGFR and DP12. Scale bar, 200 nm. Representative images. (E) Average number of nanoparticles per µm² (mean ⁺/− SD) for 550 pM (purple), 2.8 nM FGF2-NP (green). Labelling was strongly reduced in the presence of 50 µg/mL of DP12 (grey) for both concentrations of FGF2-NP. Little labelling was also observed when 550 pM of FGF2-NP was added to the cell with an excess of unlabelled FGF2 protein (50 µM) in the absence (white) or in the presence (stripped) of DP12. The number of photomicrographs of 1.578 µm per 1.578 µm or 1.578 µm per 2.1 µm analysed were 69 for 550 pM FGF2-NP, 65 for 550 pM FGF2-NP in the presence of DP12, 63 for 550 pM FGF2-NP in the presence of unlabelled FGF2, 57 for 550 pM FGF2-NP in the presence of DP12 and unlabelled FGF2, and 27 for 2.8 nM FGF2-NP and 116 for 2.8 nM FGF2-NP in the presence of DP12. Non-parametric Kolmogorov-Smirnov performed on the data gave the following <i>p</i> values: 550 pM FGF2-NP against 550 pM FGF2-NP with DP12, <i>p</i> = 0; 550 pM FGF2-NP against 550 pM FGF2-NP with excess FGF2, <i>p</i> = 0; 550 pM FGF2-NP against 550 pM FGF2-NP with DP12 and excess FGF2, <i>p</i> = 0; 550 pM FGF2-NP against 2.8 nM FGF2-NP, <i>p</i> = 2.12883E⁻⁹ all 2.8 nM FGF2-NP against 2.8 nM FGF2-NP with DP12, <i>p</i> = 2.22045E⁻¹⁶. (F) FGF2-NP clustering at 550 pM (purple) and 2.8 nM (green) was characterised by K-function analysis (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001361#s3" target="_blank">Materials and Methods</a>). 24 and 27 photomicrographs of 1.578 µm per 1.578 µm were analysed, respectively. Values of L(r)-r above the 99% confidence interval (CI) (black) indicate significant clustering within the defined <i>x</i>-axis radius values (<i>r</i>). Clustering of FGF2-NP was observed at 550 pM and its extent increased with FGF2-NP concentration.</p

An individual FGF2 undergoes several modes of diffusion.

<p>Representative trajectories of individual FGF2-NP in the pericellular matrix of Rama 27 fibroblast cells. FGF2-NP (22 pM) was incubated with living (A and B) or fixed (C, D, and E) Rama 27 fibroblasts before washes and PHI tracking. Representative trajectories of individual FGF2-NP are shown with their duration, in seconds, given using the same colour code used to trace the trajectory. (A and C) The trajectories were superimposed on the corresponding image acquired before the tracking acquisition. Scale and position axes are shown. In (B) all trajectories shown were acquired within the same area of the same cell. (E) Zoom in on three trajectories of (D). Note that the two trajectories denoted with an * correspond to two different FGF2 molecules (in green and purple) which were at the same location in the pericellular matrix, but at several minutes of interval and then followed the same path. (B, D, and E). Scale bars are shown on the image. The time course of the two FGF2 ligand trajectories within a dashed red oval in (A) and (D) corresponds to Movies S1 and S3 given in the Supporting Information section.</p

FGF2-NP stimulates DNA synthesis and the phosphorylation of FRS2 and p42/44^MAPK to the same extent as free FGF2.

<p>(A) DNA synthesis was determined in serum-starved Rama 27 fibroblasts by the incorporation of [³H] thymidine into DNA 18 h after the addition of growth factor (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001361#s3" target="_blank">Materials and Methods</a>), as follows: BSA, negative control with no growth factor; FGF2 (55 pM final) or FGF2-NP (55 pM) in the presence (+) or not (−) of 10 µg/mL heparin-derived dodecasaccharide (DP12). The results are the mean ± SD of triplicate wells of two experiments (<i>n</i> = 6). Student's <i>t</i> test was performed to compare the values. The <i>t</i> values (Prob>|<i>t</i>|) for BSA against the four conditions tested in the presence of FGF2 are shown in parenthesis on the top of the corresponding bar graph. Significant differences are observed. No significant difference was observed in between the four conditions of FGF2 stimulation (|<i>t</i>|>0.05). (B) Serum-starved Rama 27 fibroblasts were stimulated with 55 pM FGF2 or 55 pM FGF2-NP for 10 min or 40 min in the presence (+) or not (−) of 10 µg/mL DP12. The Tyr¹⁹⁶ phosphorylated form of FRS2 and the doubly phosphorylated Thr^183/202/Tyr^185/204 forms of p42/44^MAPK were detected using appropriate antibodies. The same blot was re-probed with anti-actin to show the level of loading of the gels. BSA, negative control with no growth factor added to the cells.</p

Mechanism of Electrolyte-Induced Brightening in Single-Wall Carbon Nanotubes

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

Specific binding of FGF2-NP to living and fixed cells, as revealed by photothermal heterodyne microscopy (PHI).

<p>(A, B) FGF2-NP (22 pM) was incubated for 30 min with Rama 27 fibroblasts before washes and image acquisition by PHI. (A) Image of 100×100 µm of living cells. The <i>x</i>- and <i>y</i>-axes are in µm. Nucleus is shown (white arrow). (B) Zoom in of a 10×10 µm area of (A). The <i>x</i>- and <i>y</i>-axes, in µm, giving the corresponding position in panel (A). Clear labelling was observed. (C) 22 pM TrisNiNTA-NP, not conjugated to FGF2, were used to determine non-specific binding. No labelling with nanoparticles was observed. However, some mitochondria (white arrows), which can give a signal in PHI, were observed. The signal arising from mitochondria is easily distinguishable from the signal of gold nanoparticles, notably because it bleaches <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001361#pbio.1001361-Lasne2" target="_blank">[46]</a>. (D) Living cells were incubated with 220 pM FGF2-NP in the presence of 50 µg/mL of DP12 for 30 min. In this condition, FGF2-NP binding to the HS of the pericellular matrix was abolished, but the FGF2-NP still bound FGFR. The labelling that was observed, therefore, corresponded to FGF2-NP bound to FGFR. Note that when binding of FGF2-NP to HS was abolished no labelling was observed in many of the 10×10 µm images. (E) Fixed cells were incubated with 22 pM FGF2-NP in the presence of 50 µM of unlabelled FGF2 for 30 min. In this condition, labelling was strongly reduced due to the competition between the FGF2-NP and the large excess of unlabelled FGF2. (F) Fixed cells were incubated with 22 pM FGF2-NP in the presence of unlabelled FGF2 (50 µM) and DP12 (50 µg/mL) for 30 min. In this condition, almost no labelling was observed. (G) Fixed cells were incubated with 440 pM FGF2-NP for 30 min, showing very strong labelling (52%±18 pixels labelled, mean ± SD, <i>n</i> = 28 images of 16 µm²). (H) Fixed cells incubated with heparinases I, II, and III overnight prior to incubation with 440 pM FGF2-NP for 30 min show greatly reduced labelling (6%±2.8 pixels labelled, mean ± SD, <i>N</i> = 19 images of 16 µm²). The dotted line in the upper left-hand corner indicates a background signal from a mitochondrion. Such areas were avoided for the analysis. (I) Cells in SDM were incubated overnight with chondroitinase and then fixed and incubated with 440 pM FGF2-NP. The strong labelling (49%±14 of pixels labelled, mean ± SD, <i>N</i> = 20 images of 16 µm²) is indistinguishable from the untreated control in panel G.</p

Heterogeneous diffusive behaviour of individual FGF2 in the pericellular matrix.

<p>(A) Plot of displacement (µm²) against distance travelled (µm) for FGF2-NP trajectories shown in (B) (analysis window of 12 points, 0.504 s). Five groups were defined that discriminate the different diffusive behaviours of the FGF2-NP (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001361#s3" target="_blank">Materials and Methods</a>). Group 1 (Black) immobile/high confinement (fitted according to the calculated parameter for NP embedded in thin film of polyvinyl alcohol on a glass coverslip); Group 2 (grey) confinement; Group 3 (green) simple diffusion; Group 4 (magenta) slow directed diffusion; Group 5 (blue) fast, directed diffusion (only observed in living cells). (B and C) Representative FGF2-NP trajectories at the surface of living (B) or fixed (C) Rama 27 fibroblast cells colour-coded according to the five diffusion groups defined in (A). Duration and scale bar are given on the figure.</p