Sergey Yakovlev

Effect of Morphology of Nanoscale Hydrated Channels on Proton Conductivity in Block Copolymer Electrolyte Membranes

Hydrated membranes with cocontinuous hydrophilic and hydrophobic phases are needed to transport protons in hydrogen fuel cells. Herein we study the water uptake and proton conductivity of a model fuel cell membrane comprising a triblock copolymer, polystyrenesulfonate-block-polyethylene-block-polystyrenesulfonate (S-SES), as a function of water activity in both humid air and liquid water. We demonstrate that the water uptake and proton conductivity of S-SES membranes equilibrated in liquid water are fundamentally different from values obtained when they were equilibrated in humid air. The morphological underpinnings of our observations were determined by synchrotron small-angle X-ray scattering and cryogenic scanning transmission electron microscopy. A discontinuous increase in conductivity when nearly saturated humid air is replaced with liquid water coincides with the emergence of heterogeneity in the hydrated channels: a water-rich layer is sandwiched between two polymer-rich brushes. While the possibility of obtaining heterogeneous hydrated channels in polymer electrolyte membranes has been discussed extensively, to our knowledge, this is the first time that direct evidence for the formation of water-rich subdomains is presented.

Quantitative nanoscale water mapping in frozen-hydrated skin by low-loss electron energy-loss spectroscopy

Spatially resolved low-loss electron energy-loss spectroscopy (EELS) is a powerful method to quantitatively determine the water distribution in frozen-hydrated biological materials at high spatial resolution. However, hydrated tissue, particularly its hydrophilic protein-rich component, is very sensitive to electron radiation. This sensitivity has traditionally limited the achievable spatial resolution because of the relatively high noise associated with low-dose data acquisition. We show that the damage caused by high-dose data acquisition affects the accuracy of a multiple-least-squares (MLS) compositional analysis because of inaccuracies in the reference spectrum used to represent the protein. Higher spatial resolution combined with more accurate compositional analysis can be achieved if a reference spectrum is used that better represents the electron-beam-damaged protein component under frozen-hydrated conditions rather than one separately collected from dry protein under low-dose conditions. We thus introduce a method to extract the best-fitting protein reference spectrum from an experimental spectrum dataset. This method can be used when the MLS-fitting problem is sufficiently constrained so that the only unknown is the reference spectrum for the protein component. We apply this approach to map the distribution of water in cryo-sections obtained from frozen-hydrated tissue of porcine skin. The raw spectral data were collected at doses up to 10(5)e/nm(2) despite the fact that observable damage begins at doses as low as 10(3)e/nm(2). The resulting spatial resolution of 10nm is 5-10 times better than that in previous studies of frozen-hydrated tissue and is sufficient to resolve sub-cellular water fluctuations as well as the inter-cellular lipid-rich regions of skin where water-mediated processes are believed to play a significant role in the phenotype of keratinocytes in the stratum corneum.

Electron tomography of cryo-immobilized plant tissue: a novel approach to studying 3D macromolecular architecture of mature plant cell walls in situ.

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.

Specimen thickness dependence of hydrogen evolution during cryo-transmission electron microscopy of hydrated soft materials.

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm−2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.

Direct imaging of nanoscale acidic clusters in a polymer electrolyte membrane.

One of the factors hindering the development of technologies that rely on the use of proton-conducting polyelectrolyte membranes is the lack of control over the membrane morphology on the nanoscale. Of particular importance is the rearrangement and clustering of acidic groups, which may seriously degrade the electrical properties. Although electron microscopy is capable of imaging the morphology of the clusters, images of unmodified membranes with sufficient quality to discriminate between different proposed cluster morphology models have not been presented. Here we show the first determination of the cluster size distribution in a model polymer electrolyte membrane from electron micrographs of individual acidic clusters. Imaging of the sulfur-rich clusters by dark-field microscopy was facilitated by the spontaneous formation of thin, cluster-containing layers on the top and bottom surfaces of free-standing films with a thickness of ~35 nm.

Insights on the Study of Nafion Nanoscale Morphology by Transmission Electron Microscopy

Nafion is one of the most common materials used for polyelectrolyte membranes and is the standard to which novel materials are compared. In spite of great interest in Nafion’s nanostructure, it is still a subject of controversy. While multiple research efforts have addressed Nafion’s morphology with Transmission Electron Microscopy, the results of these efforts have often been inconsistent and cannot satisfactorily describe the membrane structure. One of the reasons for differences in the reported results is the lack of sufficient control over the damage caused by electron beam irradiation. In this work, we describe some aspects of damage in the material that have a strong influence on the results. We show that irradiation causes mass loss and phase separation in the material and that the morphologies that have been observed are, in many cases, artifacts caused by damage. We study the effect of the sample temperature on damage and show that, while working at low temperature does not prevent damage and mass loss, it slows formation of damage-induced artifacts to the point where informative low-dose images of almost undamaged material may be collected. We find that charging of the sample has a substantial effect on the damage, and the importance of charge neutralization under irradiation is also seen by the large reduction of beam induced movement with the use of an objective aperture or a conductive support film. To help interpret the low-dose images, we can apply slightly higher exposures to etch away the hydrophobic phase with the electron beam and reveal the network formed by the hydrophilic phase. Energy loss spectroscopy shows evidence that fluorine removal governs the beam damage process.

Freezing in sealed capillaries for preparation of frozen hydrated sections

We have investigated the freezing of specimens in a confined volume for preparation of vitreous samples for cryosectioning. With 15% dextran as a cryoprotectant, a sample sealed in a copper tube begins to freeze into crystalline ice when plunged into liquid ethane. Crystallization rapidly causes an increase in the pressure to the point that much of the sample freezes in a vitreous state. We used synchrotron X‐ray diffraction of samples frozen with various amounts of dextran to characterize the ice phases and crystal orientation, providing insights on the freezing process. We have characterized cryosections obtained from these samples to explore the optimum amount of cryoprotectant. Images of cryosectioned bacteria frozen with various levels of cryoprotectant illustrate effects of cryoprotectant concentration.

Absence of Schroeder's Paradox in a Nanostructured Block Copolymer Electrolyte Membrane

This is a study of morphology, water uptake, and proton conductivity of a sulfonated polystyrene-block-polyethylene (PSS-PE) copolymer equilibrated in humid air with controlled relative humidity (RH), and in liquid water. Extrapolation of the domain size, water uptake, and conductivity obtained in humid air to RH = 100% allowed for an accurate comparison between the properties of PSS-PE hydrated in saturated vapor and in liquid water. We demonstrate that extrapolations of domain size and water uptake on samples equilibrated in humid air are consistent with measurements on samples equilibrated in liquid water. Small (5%) differences in proton conductivity were found in samples equilibrated in humid air and liquid water. We argue that differences in transport coefficients in disordered heterogeneous systems, particularly small differences, present no paradox whatsoever. Schroeders Paradox, wherein properties of polymers measured in saturated water vapor are different from those obtained in liquid water, is thus not observed in the PSS-PE sample.

Crystalline ice as a cryoprotectant: theoretical calculation of cooling speed in capillary tubes.

It is generally assumed that vitrification of both cells and the surrounding medium provides the best preservation of ultrastructure of biological material for study by electron microscopy. At the same time it is known that the cell cytoplasm may provide substantial cryoprotection for internal cell structure even when the medium crystallizes. Thus, vitrification of the medium is not essential for good structural preservation. By contrast, a high cooling rate is an essential factor for good cryopreservation because it limits phase separation and movement of cellular components during freezing, thus preserving the native‐like state. Here we present calculations of freezing rates that incorporate the effect of medium crystallization, using finite difference methods. We demonstrate that crystallization of the medium in capillary tubes may increase the cooling rate of suspended cells by a factor of 25–300 depending on the distance from the centre. We conclude that crystallization of the medium, for example due to low cryoprotectant content, may actually improve cryopreservation of some samples in a near native state.

Self Pressure Freezing of Caulobacter crescentus for the Preparation of Frozen Hydrated Sections

Preparation of frozen hydrated sections of biological samples such as bacteria typically requires the use of a high pressure freezing system. While such systems are commercially available, use of this type of equipment in the field, such as on-site during environmental microbiology sample collection, is very difficult and is often inconvenient in the lab. In this work we use the “self-pressurized rapid freezing” method for vitrification of Caulobacter cultures to demonstrate its suitability for preparing material for cryo-sectioning. While a similar method has been described earlier for freeze substitution [1] we adopted it for the preparation of frozen hydrated cryosections. Such a method does not require heavy equipment and therefore, is suitable for field work. The method relies on the fact that cooling water below 4oC as well as crystallization causes expansion of water (ice). Due to the low compressibility of water (ice) small expansion builds a high pressure inside of a sealed vessel. Using a sealed vessel thus eliminates the need for a high pressure freezing machine.