Correction to “Monte Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in Single-Molecule FRET”

Correction to “Monte Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in Single-Molecule FRET

Rotational and Translational Diffusion of Peptide-Coated CdSe/CdS/ZnS Nanorods Studied by Fluorescence Correlation Spectroscopy

CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (Dtrans and Drot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, Drot ∼ L-3 versus Dtrans ∼ L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that Drot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems

Far-Field Super-resolution Detection of Plasmonic Near-Fields

We demonstrate a far-field single molecule super-resolution method that maps plasmonic near-fields. The method is largely invariant to fluorescence quenching (arising from probe proximity to a metal), has reduced point-spread-function distortion compared to fluorescent dyes (arising from strong coupling to nanoscopic metallic features), and has a large dynamic range (of 2 orders of magnitude) allowing mapping of plasmonic field-enhancements regions. The method takes advantage of the sensitivity of quantum dot (QD) stochastic blinking to plasmonic near-fields. The modulation of the blinking characteristics thus provides an indirect measure of the local field strength. Since QD blinking can be monitored in the far-field, the method can measure localized plasmonic near-fields at high throughput using a simple far-field optical setup. Using this method, propagation lengths and penetration depths were mapped-out for silver nanowires of different diameters and for different dielectric environments, with a spatial accuracy of ∼15 nm. We initially use sparse sampling to ensure single molecule localization for accurate characterization of the plasmonic near-field with plans to increase density of emitters in further studies. The measured propagation lengths and penetration depths values agree well with Maxwell finite-difference time-domain calculations and with published literature values. This method offers advantages such as low cost, high throughput, and superresolved mapping of localized plasmonic fields at high sensitivity and fidelity

Comparison of Photophysical and Colloidal Properties of Biocompatible Semiconductor Nanocrystals Using Fluorescence Correlation Spectroscopy

A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs

Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides

Synthetic phytochelatin-related peptides are used as an organic coat on the surface of colloidal CdSe/ZnS semiconductor nanocrystals synthesized from hydrophobic coordinating trioctyl phosphine oxide (TOPO) solvents. The peptides are designed to bind to the nanocrystals via a C-terminal adhesive domain. This adhesive domain, composed of multiple repeats of cysteines pairs flanked by hydrophobic 3-cyclohexylalanines, is followed by a flexible hydrophilic linker domain to which various bio-affinity tags can be attached. This surface coating chemistry results in small, buffer soluble, monodisperse peptide-coated nanoparticles with high colloidal stability and ensemble photophysical properties similar to those of TOPO-coated nanocrystals. Various peptide coatings are used to modulate the nanocrystal surface properties and to bioactivate the nanoparticles. CdSe/ZnS nanocrystals coated with biotinylated peptides efficiently bind to streptavidin and are specifically targeted to GPI-anchored avidin-CD14 chimeric proteins expressed on the membranes of live HeLa cells. This peptide coating surface chemistry provides a novel approach for the production of biocompatible photoluminescent nanocrystal probes

Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides

Synthetic phytochelatin-related peptides are used as an organic coat on the surface of colloidal CdSe/ZnS semiconductor nanocrystals synthesized from hydrophobic coordinating trioctyl phosphine oxide (TOPO) solvents. The peptides are designed to bind to the nanocrystals via a C-terminal adhesive domain. This adhesive domain, composed of multiple repeats of cysteines pairs flanked by hydrophobic 3-cyclohexylalanines, is followed by a flexible hydrophilic linker domain to which various bio-affinity tags can be attached. This surface coating chemistry results in small, buffer soluble, monodisperse peptide-coated nanoparticles with high colloidal stability and ensemble photophysical properties similar to those of TOPO-coated nanocrystals. Various peptide coatings are used to modulate the nanocrystal surface properties and to bioactivate the nanoparticles. CdSe/ZnS nanocrystals coated with biotinylated peptides efficiently bind to streptavidin and are specifically targeted to GPI-anchored avidin-CD14 chimeric proteins expressed on the membranes of live HeLa cells. This peptide coating surface chemistry provides a novel approach for the production of biocompatible photoluminescent nanocrystal probes

Photobleaching Pathways in Single-Molecule FRET Experiments

To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently developed single-molecule and ensemble spectroscopies and doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways, which place fundamental limitations on sm-FRET experiments. We find that:  (i) acceptor photobleaching scales with FRET efficiency, (ii) acceptor photobleaching is enhanced under picosecond-pulsed (vs continuous-wave) excitation, and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting the use of short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements

A Rugged Energy Landscape Mechanism for Trapping of Transmembrane Receptors during Endocytosis^†

Efficient clathrin-mediated endocytosis of transmembrane receptors requires that clathrin-coated pits retain the receptors long enough to allow vesicle formation and internalization. In many cases, however, the receptors can exhibit mean lifetimes in coated pits much shorter than the lifetime of the pit at the plasma membrane. A rugged energy landscape for binding, which produces a broad distribution of residence times, ensures a significant probability of times much greater than the mean and would allow efficient trapping of proteins in these cases. We used fluorescence correlation spectroscopy and total internal reflection microscopy to measure the kinetics of movement of a C5a receptor−yellow fluorescent protein fusion in living cells. These experiments demonstrate that clusters of trapped receptors exhibit fluctuations in fluorescence intensity that vary in time scale over 2 orders of magnitude. Most of the variation in intensity is likely due to the motion of the receptors in the plane of the plasma membrane, although it is not possible to rule out a small contribution from motion orthogonal to the plane of the membrane. The broad time scale distribution of the intensity fluctuations is consistent with a rugged energy landscape mechanism for trapping of the receptors. This mechanism, which allows efficient trapping to coexist with rapid exchange, may also be relevant to other biological processes involving binding in heterogeneous chemical environments

Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation

The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton−molecular orbital (X−MO) coupling might take place in these hybrid inorganic−organic composite materials

Hybrid Approach to the Synthesis of Highly Luminescent CdTe/ZnS and CdHgTe/ZnS Nanocrystals

We report the synthesis of highly luminescent CdTe/ZnS and CdHgTe/ZnS core/shell semiconductor nanocrystals (NCs). A hybrid of two synthesis routes leads to novel nanocrystal compositions and small core/shell sizes (4−5 nm) that emit in the far-red and near-infrared regions. These particles exhibit higher resistance to oxidation and photobleaching, have high quantum yields, and could be used for biological labeling and imaging