Dependence on material choice of degradation of organic solar cells following exposure to humid air.

Electron microscopy has been used to study the degradation of organic solar cells when exposed to humid air. Devices with various different combinations of commonly used organic solar cell hole transport layers and cathode materials have been investigated. In this way the ingress of water and the effect it has on devices could be studied. It was found that calcium and aluminum in the cathode both react with water, causing voids and delamination within the device. The use of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) was found to increase the degradation by easing water ingress into the device. Replacing these materials removed these degradation features. © 2015 The Authors. Journal of Polymer Science Part B: Polymer Physics published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 216-224.

Structure of Spherulites in Insulin, ß-Lactoglobulin, and Amyloid ß.

Under denaturing conditions such as low pH and elevated temperatures, proteins in vitro can misfold and aggregate to form long rigid rods called amyloid fibrils; further self-assembly can lead to larger structures termed spherulites. Both of these aggregates resemble amyloid tangles and plaques associated with Alzheimer's disease in vivo. The ability to form such aggregates in a multitude of different proteins suggests that it is a generic ability in their mechanism to form. Little is known about the structure of these large spherulites ranging from 5 to 100 microns and whether they can reproducibly form in amyloid ß (1-40) (Aß40), a 40-amino acid residue peptide, which is one of the major components of Alzheimer's amyloid deposits. Here, we show that spherulites can readily form in Aß40 under certain monomerization and denaturing conditions. Using polarized and nonpolarized Raman spectroscopy, we analyzed the secondary structure of spherulites formed from three different proteins: insulin, ß-lactoglobulin (BLG), and Aß40. Visually, these spherulites have a characteristic "Maltese Cross" structure under crossed polarizers through an optical microscope. However, our results indicate that insulin and Aß40 spherulites have similar core structures consisting mostly of random coils with radiating fibrils, whereas BLG mostly contains ß-sheets and fibrils that are likely to be spiraling from the core to the edge.

Modeling of Noisy Spindle Dynamics Reveals Separable Contributions to Achieving Correct Orientation.

The mechanisms by which the mammalian mitotic spindle is guided to a predefined orientation through microtubule-cortex interactions have recently received considerable interest, but there has been no dynamic model that describes spindle movements toward the preferred axis in human cells. Here, we develop a dynamic model based on stochastic activity of cues anisotropically positioned around the cortex of the mitotic cell and we show that the mitotic spindle does not reach equilibrium before chromosome segregation. Our model successfully captures the characteristic experimental behavior of noisy spindle rotation dynamics in human epithelial cells, including a weak underlying bias in the direction of rotation, suppression of motion close to the alignment axis, and the effect of the aspect ratio of the interphase cell shape in defining the final alignment axis. We predict that the force exerted per cue has a value that minimizes the deviation of the spindle from the predefined axis. The model has allowed us to systematically explore the parameter space around experimentally relevant configurations, and predict the mechanistic function of a number of established regulators of spindle orientation, highlighting how physical modeling of a noisy system can lead to functional biological understanding. We provide key insights into measurable parameters in live cells that can help distinguish between mechanisms of microtubule and cortical-cue interactions that jointly control the final orientation of the spindle.

Sub-nanometre resolution imaging of polymer-fullerene photovoltaic blends using energy-filtered scanning electron microscopy.

The resolution capability of the scanning electron microscope has increased immensely in recent years, and is now within the sub-nanometre range, at least for inorganic materials. An equivalent advance has not yet been achieved for imaging the morphologies of nanostructured organic materials, such as organic photovoltaic blends. Here we show that energy-selective secondary electron detection can be used to obtain high-contrast, material-specific images of an organic photovoltaic blend. We also find that we can differentiate mixed phases from pure material phases in our data. The lateral resolution demonstrated is twice that previously reported from secondary electron imaging. Our results suggest that our energy-filtered scanning electron microscopy approach will be able to make major inroads into the understanding of complex, nano-structured organic materials.

Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns.

Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a t≃¹·8 law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a t≃¹ law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t² law for radial spreading cells, and a t¹ law for lengthwise spreading cells.

Let's not forget plants.

'Many physicists see the interface with biology as an exciting place to be'. Athene Donald provides a personal perspective on working at the interface between the physical and biological sciences.

A microrheological study of hydrogel kinetics and micro-heterogeneity.

The real-time dynamic heterogeneity of the gelation process of the amino acid derivative Fmoc-tyrosine (Fmoc-Y) is studied using particle tracking microrheology. To trigger gelation, glucono-δ-lactone (GdL) is added, which gradually lowers the p H over several hours. The onset of self-assembly in the system is signified by a sharp drop in the mean-squared displacement of embedded particles, a phenomenon that is found to correlate with the p H of the system reaching the pK(a) of Fmoc-Y. The gel point is identified and found to be dependent on the GdL concentration. Analysis of embedded probe particle dynamics allows the heterogeneity of the sample to be quantified, using three metrics: the heterogeneity ratio (HR), the non-Gaussian parameter of the van Hove correlation function (N and the bin distribution of the mean-squared displacement (MSD) of single particles (f(z)). Results from the three techniques are found to be approximately comparable, with increases in heterogeneity observed in all samples for incubation times t(w) = 0-3 hours. The final heterogeneity in all samples is found to be remarkably low compared to other systems previously reported in the literature.

Microrheology and microstructure of Fmoc-derivative hydrogels.

The viscoelasticity of hydrogel networks formed from the low-molecular-weight hydrogelator Fmoc-tyrosine (Fmoc-Y) is probed using particle-tracking microrheology. Gelation is initiated by adding glucono-δ-lactone (GdL), which gradually lowers the pH with time, allowing the dynamic properties of gelation to be examined. Consecutive plots of probe particle mean square displacement (MSD) versus lag time τ are shown to be superimposable, demonstrating the formation of a self-similar hydrogel network through a percolation transition. The analysis of this superposition yields a gel time t(gel) = 43.4 ± 0.05 min and a critical relaxation exponent n(c) = 0.782 ± 0.007, which is close to the predicted value of 3/4 for semiflexible polymer networks. The generalized Stokes-Einstein relation is applied to the master curves to find the viscoelastic moduli of the critical gel over a wide frequency range, showing that the critical gel is structurally and rheologically fragile. The scaling of G'/G″ as ω(0.795±0.099) ≈ ω(3/4) at high frequencies provides further evidence for semiflexible behavior. Cryogenic scanning electron micrographs depict a loosely connected network close to the gel point with a fibrillar persistence length that is longer than the network mesh size, further indications of semiflexible behavior. The system reported here is one of a number of synthetic systems shown to exhibit semiflexible behavior and indicates the opportunity for further rheological study of other Fmoc derivatives.

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.

Automated tracking of mitotic spindle pole positions shows that LGN is required for spindle rotation but not orientation maintenance.

Spindle orientation defines the plane of cell division and, thereby, the spatial position of all daughter cells. Here, we develop a live cell microscopy-based methodology to extract spindle movements in human epithelial cell lines and study how spindles are brought to a pre-defined orientation. We show that spindles undergo two distinct regimes of movements. Spindles are first actively rotated toward the cells' long-axis and then maintained along this pre-defined axis. By quantifying spindle movements in cells depleted of LGN, we show that the first regime of rotational movements requires LGN that recruits cortical dynein. In contrast, the second regime of movements that maintains spindle orientation does not require LGN, but is sensitive to 2ME2 that suppresses microtubule dynamics. Our study sheds first insight into spatially defined spindle movement regimes in human cells, and supports the presence of LGN and dynein independent cortical anchors for astral microtubules.

Electrostatics controls the formation of amyloid superstructures in protein aggregation.

The possibility for proteins to aggregate in different superstructures, i.e. large-scale polymorphism, has been widely observed, but an understanding of the physicochemical mechanisms behind it is still out of reach. Here we present a theoretical model for the description of a generic aggregate formed from an ensemble of charged proteins. The model predicts the formation of multifractal structures with the geometry of the growth determined by the electrostatic interactions between single proteins. The model predictions are successfully verified in comparison with experimental curves for aggregate growth allowing us to reveal the mechanism of formation of such complex structures. The model is general and is able to predict aggregate morphologies occurring both in vivo and in vitro. Our findings provide a framework where the physical interactions between single proteins, the aggregate morphology, and the growth kinetics are connected into a single model in agreement with the experimental data.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM.

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and ΔmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the ΔmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type.

Factors affecting the formation of insulin amyloid spherulites.

Thermally induced amyloid aggregation of bovine insulin can produce a number of distinct aggregate morphologies. In this work amyloid spherulites were analysed using cross polarized optical microscopy and light scattering. A new semi-quantitative methodology to estimate the balance of spherulites and free fibrils is reported and, from this analysis, the effects of pH, temperature, salt, and protein concentration on spherulite formation were quantitatively determined for the first time. The number and size of spherulites measured with polarized light microscopy were related to changes in the colloidal stability of the solution and fibril nucleation times (measured by static light scattering). Importantly, changes in pH between 1.75 and 2 were found to result in a dramatic decrease in the spherulite radii, which were related to differences in the conformational stability of the protein. Moreover, estimates of the final spherulite volume fraction clearly indicate that amyloid spherulite formation is the dominant pathway for insulin aggregation in HCl solutions at low pH and protein concentrations below ~5 mg ml(-1), with the balance shifting towards fibrils as the concentration increases.

Imaging internal features of whole, unfixed bacteria.

Wet scanning-transmission electron microscopy (STEM) is a technique that allows high-resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique.

A micro-incubator for cell and tissue imaging.

A low-cost micro-incubator for imaging dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment that can be altered to study responses of cell monolayers for several days as well as relatively thick tissue samples and tissue-engineered epithelial tissues in experiments lasting several hours. Samples are contained in a sterile cavity closed by a gas-permeable membrane. The incubator can be positioned in any direction and used on an inverted or upright microscope. Temperature is regulated using a Peltier module controlled by a sensor positioned close to the sample, enabling compensation for any changes in temperature. Rapid changes in a sample's surrounding environment can be achieved due to the fast response of the Peltier module. These features permit monitoring of sample adaptation to induced environmental changes.

Spherulites of amyloid-beta42 in vitro and in Alzheimer's disease.

Several amyloidogenic proteins including insulin, beta-lactoglobulin, and albumin form spherulites in vitro under non-physiological conditions. These micrometer-sized, roughly spherical structures are composed of ordered arrays of amyloid fibrils in radial arrangements which, characteristically, show a typical Maltese cross pattern of light extinction under the polarizing microscope. The physiological significance of amyloid spherulites is unknown though in Alzheimer's disease, senile plaques composed primarily of beta sheets of amyloid-beta (Abeta)42 have, very occasionally, been shown to give a Maltese cross pattern of light extinction under crossed polarizers. Herein we describe the first observation of the formation in vitro of spherulites of Abeta42. They were formed under near-physiological conditions in which the beta sheet conformation of pre-formed aggregates of Abeta42 had been abolished following the addition of an excess of copper. Incubation of these preparations at 37 degrees C for up to 9 months resulted in the formation of globular structures, 5-20 microm in diameter, which exhibited a Maltese cross pattern of light extinction typical of spherulites. Near-identical spherulitic structures were also observed in abundance in 30 microm thick sections of Alzheimer's disease brain tissue. Synchrotron x-ray fluorescence showed that the location of these spherulites in AD tissue coincided with locally elevated concentrations of tissue copper. The formation in vitro of spherulites of Abeta42 which morphologically appeared analogous to spherulitic structures observed in vivo strongly supports the hypothesis that spherulites and senile plaques in AD tissue are one and the same structures and that their ultimate formation may involve copper.

Cracking of drying latex films: an ESEM experiment.

In this study environmental scanning electron microscopy was used to observe the cracking of drying latex films below their glass-transition temperature. By controlling the relative humidity so that it decreases linearly with time, a critical level of humidity at which cracking occurs can be determined and this is measured as a function of film thickness. It was found that the cracking humidity decreases with increases in film thickness for thicknesses in the range of 30 to 100 mum and then remains almost unchanged. A scaling argument can be used to fit the data very well and indicates that cracking occurs as soon as the entire film is consolidated into close packing.