Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots.

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery.

Exploring in vivo cholesterol-mediated interactions between activated EGF receptors in plasma membrane with single-molecule optical tracking.

BACKGROUND: The first step in many cellular signaling processes occurs at various types of receptors in the plasma membrane. Membrane cholesterol can alter these signaling pathways of living cells. However, the process in which the interaction of activated receptors is modulated by cholesterol remains unclear. METHODS: In this study, we measured single-molecule optical trajectories of epidermal growth factor receptors moving in the plasma membranes of two cancerous cell lines and one normal endothelial cell line. A stochastic model was developed and applied to identify critical information from single-molecule trajectories. RESULTS: We discovered that unliganded epidermal growth factor receptors may reside nearby cholesterol-riched regions of the plasma membrane and can move into these lipid domains when subjected to ligand binding. The amount of membrane cholesterol considerably affects the stability of correlated motion of activated epidermal growth factor receptors. CONCLUSIONS: Our results provide single-molecule evidence of membrane cholesterol in regulating signaling receptors. Because the three cell lines used for this study are quite diverse, our results may be useful to shed light on the mechanism of cholesterol-mediated interaction between activated receptors in live cells.

Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo.

Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo.

Unraveling the impact of lipid domains on the dimerization processes of single-molecule EGFRs of live cells.

Epidermal growth factor receptor (EGFR/ErbB1) is a transmembrane protein that can drive cell growth and survival via the ligand-induced dimerization of receptors. Because dimerization is a common mechanism for signal transduction, it is important to improve our understanding of how the dimerization process and membrane structure regulate signal transduction. In this study, we examined the effect of lipid nanodomains on the dimerization process of EGFR molecules. We discovered that after ligand binding, EGFR molecules may move into lipid nanodomains. The lipid nanodomains surrounding two liganded EGFRs can merge during their correlated motion. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures both the sensitivity of single-molecule processes and statistic accuracy of data analysis, providing insight into the connection between the mobile clustering process of receptors and the hierarchical structure of plasma membrane.

Energetic modeling and single-molecule verification of dynamic regulation on receptor complexes by actin corrals and lipid raft domains.

We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane of a living cell. Simulation results are presented to elaborate the confinement effects from actin corrals and protein-induced lipid domains. Single-molecule tracking data of epidermal growth factor receptors (EGFR) acquired on live HeLa cells agree with the simulation results and the mechanism that controls the diffusion of single-molecule receptors is clarified. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

Quantum control study of ultrafast optical responses in semiconductor quantum dot devices.

Two quantum control spectroscopic techniques were applied to study InAs quantum dot (QD) devices, which contain different strain-reducing layers. By adaptively control light matter interaction, a delayed resonant response from the InAs QDs was found to be encoded into the optimal phase profile of ultrafast optical pulse used. We verified the delayed resonant response to originate from excitons coupled to acoustic phonons of InAs QDs with two-dimensional coherent spectroscopy. Our study yields valuable dynamical information that can deepen our understanding of the coherent coupling process of exciton in the quantum-confined systems.

Deciphering the catalysis-associated conformational changes of human adenylate kinase 1 with single-molecule spectroscopy.

Human adenylate kinase isoenzyme 1 (AK1) is the key enzyme in maintaining the cellular energy homeostasis. The catalysis-associated conformational changes of AK1 involve large-amplitude rearrangements. To decipher the conformational changes of AK1 at the single-molecule level, we tagged AK1 with two identical fluorophores, one near the substrate-binding site and the other at the boundary of the core domain. We found that magnesium ion binding to AK1 increases the structural heterogeneity of AK1, whereas ADP binding reduces the structural heterogeneity. We exploited the hidden Markov model to extract the underlying catalysis-associated conformational dynamics and determined thermodynamic parameters of the multiple catalytic pathways. The third-order correlation difference calculated from photon fluctuation traces reveals the irreversible nature of the conformational motions, suggesting that single-molecule AK1 is in a nonequilibrium steady state. This discovery offers a fresh viewpoint to look into the molecular mechanisms of cellular biochemistry.

Dynamic regulation on energy landscape evolution of single-molecule protein by conformational fluctuation.

We formalize a theory to help explore the effect of conformational fluctuation on the energy landscape evolution of single-molecule protein. Using this formalization, we investigate the photon emission from single photoactivated fluorescent protein. A bimodal regulation on the energy landscape evolution was discovered, and its origin was attributed to slow conformational fluctuations of the protein matrix.

Self assembled liquid crystal polymers on photo-irradiated alignment surfaces for tailoring response properties of liquid crystal molecules.

A liquid crystal polymer (LCP) self-assembled on a photoirradiated substrate can modify the viscoelastic response of liquid crystal medium on the substrate. Sum-frequency vibrational spectroscopy shows that the phenyl groups of LCP are oriented epitaxially with layer thickness and an in-plane alignment order much higher than that at the photoirradiated surface can be yielded. The liquid crystal molecules confined between the LCP-coated substrates reveals a stronger correlation among the thermally excited fluctuation modes. Our finding can be used to tailor the boundary forces on alignment substrates and to optimize the device performance.

Impact of electric quadrupolar coupling on the optical response of an array of nano-objects.

Intra- and inter-particle coupling effects are important but have not been properly taken into account in modeling the optical response of an array of nano-objects. In this paper, we present a method to analyze the impact of electric quadrupolar coupling on the optical response of a layer of silver nanorods fabricated with oblique-angle deposition (OAD). Our technique can render the non-locally coupled nano-objects into an array of coarse-grained induced charges. The retrieved polarizability tensor of the silver nanorods exhibits non-zero off-diagonal components, revealing information about the array structure and inter-particle quadrupolar couplings. Thus, a more transparent picture of the correlation between the function and structure of an optical metamaterial can be yielded.

Generation and spectral manipulation of coherent terahertz radiation with two-stage optical rectification.

We propose and experimentally demonstrate the generation of single-cycle terahertz radiation with two-stage optical rectification in GaSe crystals. By adjusting the time delay between the pump pulses employed to excite the two stages, the terahertz radiation from the second GaSe crystal can constructively superpose with the terahertz field injected from the first stage. The high mutual coherence between the two terahertz radiation fields is ensured with the coherent optical rectification process and can be further used to synthesize a desired spectral profile of coherent THz radiation. The technique is also potentially useful for generating high-power single-cycle terahertz pulses, usually limited by the pulse walk-off effect of the nonlinear optical crystal used.

Probing field-induced submolecular motions in a ferroelectric liquid crystal mixture with time-resolved two-dimensional infrared spectroscopy.

Time-resolved two-dimensional infrared (2D IR) spectroscopy has been applied to analyse an electro-optic switching ferroelectric liquid crystal (FLC) mixture. The 2D IR correlation technique clearly shows that the Goldstone mode in the SmC* phase is suppressed by an applied electric field. The field-induced reorientation process initiates from intramolecular motions in about 10 µs. The intramolecular motions then propagate from the molecular segments attached to the same molecule to those fragments on other surrounding molecules. During the field-induced switching, the IR dipoles undergo a collective reorientation but with hindered rotation about the molecular long axis.

Erbium doped GaSe crystal for mid-IR applications.

We reported a type-I difference-frequency generator (DFG), based on erbium doped GaSe (Er:GaSe) crystals as a coherent infrared source tunable from 2.4 mum to 28 mum. The two mixing beams used for the DFG are a tunable near infrared output (1.1-1.8 mum) from an optical parametric amplifier (OPA) and the fundamental beam of a picosecond Nd:YAG laser at 1.064 mum. The system can produce a maximum output pulse energy of 5 microJ at wavelength of 3.5 microm, corresponding to a photon conversion efficiency of 8% at a pump intensity of 1.7 GW/cm(2). The nonlinear coefficient (d(eff)) of 0.5 atom % erbium doped GaSe crystal was found to be 55.3 pm/V or 24 % higher than that of a pure GaSe crystal. The improvement of d(eff) is attributed to the substitutive and interstitial doping of Er ion in GaSe unit cell. The optical properties of GaSe influenced by the erbium doping are also presented.

Generation properties of coherent infrared radiation in the optical absorption region of GaSe crystal.

We report a study of the effect of optical absorption on generation of coherent infrared radiation from mid-IR to THz region from GaSe crystal. The infrared-active modes of epsilon-GaSe crystal at 236 cm(-1) and 214 cm(-1) were found to be responsible for the observed optical dispersion and infrared absorption edge. Based upon phase matching characteristics of GaSe for difference-frequency generation (DFG), new Sellmeier equations of GaSe were proposed. The output THz power variation with wavelength can be properly explained with a decrease of parametric gain and the spectral profile of absorption coefficient of GaSe. The adverse effect of infrared absorption on (DFG) process can partially be compensated by doping GaSe crystal with erbium ions.

Pulse retrieval from interferometric autocorrelation measurement by use of the population-split genetic algorithm.

The population-split genetic algorithm (PSGA) was successfully applied to retrieve femtosecond optical fields from interferometric autocorrelation traces. PSGA strikes a balance between diversity and the size of population in the genetic algorithm. As a result, PSGA is less likely prematurely converging to sub-optimal solutions. Theoretical and experimental studies indicate that the PSGA can yield more accurate results in shorter time compared with conventional genetic algorithm and the iterative method. compared with conventional genetic algorithm and iterative method.

Diode-pumped passively mode-locked multiwatt Nd:GdVO4 laser with a saturable Bragg reflector.

We report a first demonstration, to our knowledge, of a cw passively mode-locked Nd:GdVO4 laser (k = 1063 nm). A relaxed saturable Bragg reflector was used. The laser generates pulses of 9.2 ps at a repetition rate of 119 MHz. As much as 5.4 W of average power was realized with a slope efficiency of 25.7%.