Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations.

Self-pressurised rapid freezing (SPRF) has been proposed as a simple alternative to traditional high-pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 MPa, however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fibre-optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure build-up during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulphoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt% are exceeded. Since a trade-off between pressure and DMSO concentration represents an impasse with regard to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fibre-facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two-chamber designs. Lay version of abstract for 'Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations' Anyone is familiar with pipes bursting in winter because the volume of ice is greater than that of liquid water. Less well known is the fact that inside a thick-walled container, sealed and devoid of air bubbles, this pressure build-up will allow a fraction of water to remain unfrozen if the sample is also cooled sufficiently rapidly far below the freezing point. This phenomenon has already been harnessed for specimen preparation in microscopy, where low temperatures are useful to immobilise the sample, but harmful if ice formation occurs. However, specimen preparation cannot always rely on this pressure-based effect alone, but sometimes requires addition of chemicals to inhibit ice formation. Not enough is known directly about how these chemicals affect pressure build-up: Indeed, rapid cooling below the freezing point is only possible for small sample volumes, typically placed inside sealed capillaries, so that space is generally insufficient to accommodate a pressure sensor. By means of a compact sensor, based on an optical fibre, laser and spectrometer, we present the first direct assessment of pressure inside sealed capillaries. We show that addition of chemicals reduces pressure build-up and present a two-chambered capillary to circumvent the resulting trade-off. Also, we present evidence showing that the two-chambered capillary design can avoid ice formation more readily than a single-chambered one.

Lifting restrictions on coherence loss when characterizing non-transparent hypersonic phononic crystals.

Hypersonic phononic bandgap structures confine acoustic vibrations whose wavelength is commensurate with that of light, and have been studied using either time- or frequency-domain optical spectroscopy. Pulsed pump-probe lasers are the preferred instruments for characterizing periodic multilayer stacks from common vacuum deposition techniques, but the detection mechanism requires the injected sound wave to maintain coherence during propagation. Beyond acoustic Bragg mirrors, frequency-domain studies using a tandem Fabry-Perot interferometer (TFPI) find dispersions of two- and three-dimensional phononic crystals (PnCs) even for highly disordered samples, but with the caveat that PnCs must be transparent. Here, we demonstrate a hybrid technique for overcoming the limitations that time- and frequency-domain approaches exhibit separately. Accordingly, we inject coherent phonons into a non-transparent PnC using a pulsed laser and acquire the acoustic transmission spectrum on a TFPI, where pumped appear alongside spontaneously excited (i.e. incoherent) phonons. Choosing a metallic Bragg mirror for illustration, we determine the bandgap and compare with conventional time-domain spectroscopy, finding resolution of the hybrid approach to match that of a state-of-the-art asynchronous optical sampling setup. Thus, the hybrid pump-probe technique retains key performance features of the established one and going forward will likely be preferred for disordered samples.

Large T g Shift in Hybrid Bragg Stacks through Interfacial Slowdown.

Studies of glass transition under confinement frequently employ supported polymer thin films, which are known to exhibit different transition temperature T g close to and far from the interface. Various techniques can selectively probe interfaces, however, often at the expense of sample designs very specific to a single experiment. Here, we show how to translate results on confined thin film T g to a "nacre-mimetic" clay/polymer Bragg stack, where periodicity allows to limit and tune the number of polymer layers to either one or two. Exceptional lattice coherence multiplies signal manifold, allowing for interface studies with both standard T g and broadband dynamic measurements. For the monolayer, we not only observe a dramatic increase in T g (∼ 100 K) but also use X-ray photon correlation spectroscopy (XPCS) to probe platelet dynamics, originating from interfacial slowdown. This is confirmed from the bilayer, which comprises both "bulk-like" and clay/polymer interface contributions, as manifested in two distinct T g processes. Because the platelet dynamics of monolayers and bilayers are similar, while the segmental dynamics of the latter are found to be much faster, we conclude that XPCS is sensitive to the clay/polymer interface. Thus, large T g shifts can be engineered and studied once lattice spacing approaches interfacial layer dimensions.

Flash Brillouin Scattering: A Confocal Technique for Measuring Glass Transitions at High Scan Rates.

Glass transition temperatures T g are most commonly measured by differential scanning calorimetry, a method that has been extended to the flash scanning calorimetry (FSC) regime by reducing sample volumes. However, significant manual preparation effort can render FSC impractical for, e.g., local probing of spatially heterogeneous specimens. Another strategy can be to select a small volume by focusing down a laser beam, where Brillouin Light Scattering (BLS) is a proven method for confocal T g measurement. Here, we introduce Flash Brillouin Scattering, which extends BLS to fast scan rates, achieved by periodically heating the probed region with an infrared laser. For comparison with conventional BLS, we first characterize T g of pure glycerol, and show how rapid quenching produces a less packed glass with downshifted sound velocity. We then turn toward its aqueous solutions, which crystallize too fast for a nonflash approach, and demonstrate scan rates in excess of 105 K/s. These results are of interest not only because glycerol is a model system for hydrogen-bonded glass formers, but also because of its applications as a cryoprotectant for frozen biological samples. Light scattering studies of the latter, currently limited to cryo-Raman spectroscopy, are likely to be complemented by the technique introduced here.

Tunable Thermoelastic Anisotropy in Hybrid Bragg Stacks with Extreme Polymer Confinement.

Controlling thermomechanical anisotropy is important for emerging heat management applications such as thermal interface and electronic packaging materials. Whereas many studies report on thermal transport in anisotropic nanocomposite materials, a fundamental understanding of the interplay between mechanical and thermal properties is missing, due to the lack of measurements of direction-dependent mechanical properties. In this work, exceptionally coherent and transparent hybrid Bragg stacks made of strictly alternating mica-type nanosheets (synthetic hectorite) and polymer layers (polyvinylpyrrolidone) were fabricated at large scale. Distinct from ordinary nanocomposites, these stacks display long-range periodicity, which is tunable down to angstrom precision. A large thermal transport anisotropy (up to 38) is consequently observed, with the high in-plane thermal conductivity (up to 5.7 W m-1 K-1 ) exhibiting an effective medium behavior. The unique hybrid material combined with advanced characterization techniques allows correlating the full elastic tensors to the direction-dependent thermal conductivities. We, therefore, provide a first analysis on how the direction-dependent Young's and shear moduli influence the flow of heat.