Mark A. Osborne

Brightening, Blinking, Bluing and Bleaching in the Life of a Quantum Dot: Friend or Foe?

Semiconductor nanocrystals or quantum dots (QDs) are highly photoluminescent materials with unique optical attributes that are being exploited in an ever-increasing array of applications. However, the complex surface chemistry of these finite-sized fluorophores gives rise to a number of photophysical phenomena that can complicate their use in imaging applications. Fluorescence intermittency (FI), photoluminescence enhancement (PLE) and spectral bluing are properties of QD emission that would appear, at first sight, detrimental to quantitative measurement. Fortunately, developments in rational QD synthesis and surface modification are promising to minimize the effects of these fluorescence instabilities, while applications that exploit them are now coming to the fore. We review recent experimental and theoretical studies of FI, PLE and bluing, highlighting the benefits, as well as complications, they bring to key applications.

Rotational transfer, an angular momentum model

We have re‐examined critical experiments on collision induced rotational transfer (RT) and conclude that the probability of RT is controlled by the factors that control the probability of angular momentum (AM) change. The probability of energy change seems less important in this respect. In the light of this we suggest a model for RT in which the probability of AM change is calculated directly and present a formalism for this purpose. We demonstrate that such a calculation leads to an exponential‐like fall of RT probabilities with transferred AM, a consequence of the radial dependence of the repulsive part of the intermolecular potential. Thus in this AM model, the exponential gap law has a simple physical origin. The AM model we describe may be used as the basis of an inversion routine through which it is possible to convert RT data into a probability density of the repulsive anisotropy. Through this model therefore it is possible to relate experimental RT data directly to the forces that are responsible...

A fitting law for rotational transfer rates: An angular momentum model with predictive power

We have formulated a law for state‐to‐state rotational transfer (RT) in diatomic molecules based on the angular momentum (AM) theory proposed by McCaffery et al. [J. Chem. Phys. 98, 4586 (1993)]. In this, the probability of angular momentum change in the rotor is calculated by assuming the dominant process to be the conversion of linear to angular momentum at the repulsive wall of the intermolecular potential. The result is a very simple expression containing three variable parameters, each of which has physical significance in the context of the model. Fits to known RT data are very good and suggest strongly that linear to angular momentum change is indeed the controlling process in RT. The parameters of the fit are sufficiently available to give the model predictive power. Using this formulation, RT probabilities may be calculated for an unknown system with little more than the atomic masses, bond length, and velocity distribution. We feel that this represents an important step in the development of a s...

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and by small inhibitory proteins that associate with the R1 catalytic subunit. In addition, the subcellular localization of the R2 subunit is regulated through the cell cycle and in response to DNA damage. We show that the fission yeast small RNR inhibitor Spd1 is intrinsically disordered and regulates R2 nuclear import, as predicted by its relationship to Saccharomyces cerevisiae Dif1. We demonstrate that Spd1 can interact with both R1 and R2, and show that the major restraint of RNR in vivo by Spd1 is unrelated to R2 subcellular localization. Finally, we identify a new behavior for RNR complexes that potentially provides yet another mechanism to regulate dNTP synthesis via modulation of RNR complex architecture.

Quantized momentum mechanics of inelastic and reactive collisions: the role of energy and angular momentum constraints

In addition to the quantized molecular structure that constrains the kinematics of atom-molecule collision, constraints arising from energy and angular momentum (AM) conservation strongly influence the magnitude and probability distribution of rotational transfer due to collisions. However, once the nature of these constraints is established, it is possible to predict quantitatively, using quantized momentum mechanics, the outcome of inelastic and reactive collisions. The existence of these constraints is most clearly portrayed in plots of relative velocity versus final rotational AM . These represent channel opening under the conditions: (i) energy conservation, (ii) simultaneous energy and angular momentum conservation, the latter via a specified maximum effective impact parameter and (iii) angular momentum conservation via this same is generally set to be the half bond length (HBL) of the diatomic, as suggested by experimental and theoretical considerations. Each of these conditions may constrain the rotational transfer process. The plots also give indications of conditions under which backward or forward scattering will occur and when long-lived complexes will form. For sharply defined velocity distributions, scattering angle peaks are readily calculated and these agree well with experimental data. When energy constraints dominate, the value of may be less than HBL and this reduced value may be obtained from the kinematic equations. This permits wider usage of the AM theory of rotational transfer. AM constraints effectively reduce the number of channels that appear to be accessible from energetic considerations. The plots may also be used to analyse reactive collisions and inelastic processes in polyatomic molecules for data in which the angular momentum change is directed principally along one of the molecules inertial axes.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

Dynamical angular momentum models for rotational transfer in polyatomic molecules

We propose a model for collision‐induced rotational transfer (RT) in polyatomic molecules based on the angular momentum (AM) sphere, a classical representation of the dynamical motion of the rotational AM vector in the molecular frame. The model develops further that proposed by us [AlWahabi et al., J. Chem. Soc., Faraday Trans. 85, 1003 (1989)] in which RT probabilities are related to the AM gap linking initial and final Nkakc states. The AM sphere representation embodies the full internal motion of the molecule via its effect on the inertial axes and the trajectory of the individual rotational state vectors. In this representation there is no unique AM gap for a particular transition between states of nominally well‐defined Nkakc and here we propose and test several models for obtaining the distance in AM space between initial and final trajectories. Models are evaluated from their ability to fit data on NH2–H collisions. We find that even the simplest approximations, such as shortest distance in AM spa...

Machinery of Government

A study using genome-wide epigenetic profiling has shown that heavy smoking causes reduced levels of DNA methylation in a gene that encodes a drug target for cardiovascular disease. Methodological limitations have meant that most studies of the potential links between DNA methylation and disease have so far focused on those CpGs (the most common sites of DNA methylation in humans) for which there is pre-existing reason to suspect a disease connection. Breitling and colleagues used an array-based platform for genome-wide DNA methylation profiling to study the effects of cigarette smoking, free of any a priori hypothesis about which positions might be affected. DNA methylation was measured at 27,578 CpGs in promoters across the genome in peripheral blood from 177 people, including heavy smokers, former heavy smokers and those who had never smoked. The authors identified one site — in the gene coagulation factor II receptor-like 3 (F2RL3) — that showed genome-wide significance for reduced DNA methylation in heavy smokers. This association was replicated in an independent sample of 328 people. Excitingly, despite the fact that it had not previously been implicated in the effects of smoking, F2RL3 encodes a protein that has functions which are relevant to cardiovascular disease, and is in fact a potential drug target for that condition. Although the functional impact of this difference in DNA methylation remains to be explored, this study illustrates the potential of epigenomics to provide insights into mechanisms of disease, and to suggest new avenues for clinical intervention.

Sizing Up Excitons in Core–Shell Quantum Dots via Shell-Dependent Photoluminescence Blinking

Semiconductor nanocrystals or quantum dots (QDs) are now widely used across solar cell, display, and bioimaging technologies. While advances in multishell, alloyed, and multinary core-shell QD structures have led to improved light-harvesting and photoluminescence (PL) properties of these nanomaterials, the effects that QD-capping have on the exciton dynamics that govern PL instabilities such as blinking in single-QDs is not well understood. We report experimental measurements of shell-size-dependent absorption and PL intermittency in CdSe-CdS QDs that are consistent with a modified charge-tunnelling, self-trapping (CTST) description of the exciton dynamics in these nanocrystals. By introducing an effective, core-exciton size, which accounts for delocalization of charge carriers across the QD core and shell, we show that the CTST models both the shell-depth-dependent red-shift of the QD band gap and changes in the on/off-state switching statistics that we observe in single-QD PL intensity trajectories. Further analysis of CdSe-ZnS QDs, shows how differences in shell structure and integrity affect the QD band gap and PL blinking within the CTST framework.

Direct object resolution by image subtraction: a new molecular ruler for nanometric measurements on complexed fluorophores

A technique for measuring distances between two or more fluorophores spaced in the 10-100 nm range is described. We identify a linear correlation between the intensity-amplitude in the difference-image of single molecules undergoing fluorescence fluctuations and their separation. The transform is used to map distances between coupled fluorophores.