New science exploration from XFEL: a new paradigm for structural visualisation of macromolecules

X-rays have a long-standing history as an investigative probe in the sciences, and in particular their application to the biological and biomedical sciences has provided an enormous contribution to these fields. Indeed structural biology, the study of the molecules of life at an atomic scale via macromolecular crystallography, has been a major benefactor of advances in x-ray radiation sources. Currently two major bottlenecks exist within this field, the need for well diffracting crystals and radiation damage limitations. The advent of fourth generation x-ray sources, X-ray Free-electron Lasers (XFEL) heralds a shift in the way such experiments are performed. XFELs, due to their high brilliance and ultra short (fs) pulses, hope to decouple radiation dose limitations from spatial resolution by outrunning this radiation damage in short exposures, ‘diffraction before destruction’. This thesis is concerned with exploring experimental methodologies made possible by XFELs, including establishing the experimental infrastructure required at the worlds second XFEL, SACLA, and performing initial experiments. Firstly the potential of performing gas-phase small angle x-ray scattering experiments (gSAXS) is investigated. The current need for gas-phase structural information will be presented and the experimental parameters and projected signal requirements will then be explored. The results of experiments at a synchrotron radiation source with various biomolecules will be presented. It is shown that with the current experimental set-up experiments are fundamentally limited by the signal to noise ratio (SNR) pointing to the necessity of XFEL. Secondly the application of coherent diffractive imaging (CDI) to biological systems at synchrotron and XFEL sources is explored, and the development of experimental systems at both sources is outlined. A method for combining complimentary scattering experiments at both sources is demonstrated and the results of its application to the assembly mechanism of the self-assembling, non-crystalline, macromolecule, the RNAi microsponge, are presented. The microsponge is found to have a nucleating origin leading to a core-shell like nanostructure in the fully formed molecule

GENFIRE: A generalized Fourier iterative reconstruction algorithm for high-resolution 3D imaging

Tomography has made a radical impact on diverse fields ranging from the study of 3D atomic arrangements in matter to the study of human health in medicine. Despite its very diverse applications, the core of tomography remains the same, that is, a mathematical method must be implemented to reconstruct the 3D structure of an object from a number of 2D projections. In many scientific applications, however, the number of projections that can be measured is limited due to geometric constraints, tolerable radiation dose and/or acquisition speed. Thus it becomes an important problem to obtain the best-possible reconstruction from a limited number of projections. Here, we present the mathematical implementation of a tomographic algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE). By iterating between real and reciprocal space, GENFIRE searches for a global solution that is concurrently consistent with the measured data and general physical constraints. The algorithm requires minimal human intervention and also incorporates angular refinement to reduce the tilt angle error. We demonstrate that GENFIRE can produce superior results relative to several other popular tomographic reconstruction techniques by numerical simulations, and by experimentally by reconstructing the 3D structure of a porous material and a frozen-hydrated marine cyanobacterium. Equipped with a graphical user interface, GENFIRE is freely available from our website and is expected to find broad applications across different disciplines.Comment: 18 pages, 6 figure

Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction.

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three-dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three-dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three-dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction

In situ coherent diffractive imaging

Coherent diffractive imaging (CDI) has been widely applied in the physical and biological sciences using synchrotron radiation, XFELs, high harmonic generation, electrons and optical lasers. One of CDI's important applications is to probe dynamic phenomena with high spatio-temporal resolution. Here, we report the development of a general in situ CDI method for real-time imaging of dynamic processes in solution. By introducing a time-invariant overlapping region as a real-space constraint, we show that in situ CDI can simultaneously reconstruct a time series of the complex exit wave of dynamic processes with robust and fast convergence. We validate this method using numerical simulations with coherent X-rays and performing experiments on a materials science and a biological specimen in solution with an optical laser. Our numerical simulations further indicate that in situ CDI can potentially reduce the radiation dose by more than an order of magnitude relative to conventional CDI. As coherent X-rays are under rapid development worldwide, we expect in situ CDI could be applied to probe dynamic phenomena ranging from electrochemistry, structural phase transitions, charge transfer, transport, crystal nucleation, melting and fluid dynamics to biological imaging.Comment: 19 pages, 5 figure

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features in the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe$_3$O$_4$ core encased by a 25-nm-thick fluorescent silica (SiO$_2$) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity.

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI's quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI’s quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 ± 1.0 nm and 4.0 ± 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity

Mutual fund performance: Measurement and evidence

The paper provides a critical review of empirical findings on the performance of mutual funds, mainly for the US and UK. Ex-post, there are around 0-5% of top performing UK and US equity mutual funds with truly positive-alpha performance (after fees) and around 20% of funds that have truly poor alpha performance, with about 75% of active funds which are effectively zero-alpha funds. Key drivers of relative performance are, load fees, expenses and turnover. There is little evidence of successful market timing. Evidence suggests past winner funds persist, when rebalancing is frequent (i.e., less than one year) and when using sophisticated sorting rules (e.g., Bayesian approaches) - but transactions costs (load and advisory fees) imply that economic gains to investors from winner funds may be marginal. The US evidence clearly supports the view that past loser funds remain losers. Broadly speaking results for bond mutual funds are similar to those for equity funds. Sensible advice for most investors would be to hold low cost index funds and avoid holding past 'active' loser funds. Only sophisticated investors should pursue an active ex-ante investment strategy of trying to pick winners - and then with much caution. © 2010 New York University Salomon Center and Wiley Periodicals, Inc

Reading and Ownership

First paragraph: ‘It is as easy to make sweeping statements about reading tastes as to indict a nation, and as pointless.’ This jocular remark by a librarian made in the Times in 1952 sums up the dangers and difficulties of writing the history of reading. As a field of study in the humanities it is still in its infancy and encompasses a range of different methodologies and theoretical approaches. Historians of reading are not solely interested in what people read, but also turn their attention to the why, where and how of the reading experience. Reading can be solitary, silent, secret, surreptitious; it can be oral, educative, enforced, or assertive of a collective identity. For what purposes are individuals reading? How do they actually use books and other textual material? What are the physical environments and spaces of reading? What social, educational, technological, commercial, legal, or ideological contexts underpin reading practices? Finding answers to these questions is compounded by the difficulty of locating and interpreting evidence. As Mary Hammond points out, ‘most reading acts in history remain unrecorded, unmarked or forgotten’. Available sources are wide but inchoate: diaries, letters and autobiographies; personal and oral testimonies; marginalia; and records of societies and reading groups all lend themselves more to the case-study approach than the historical survey. Statistics offer analysable data but have the effect of producing identikits rather than actual human beings. The twenty-first century affords further possibilities, and challenges, with its traces of digital reader activity, but the map is ever-changing