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.

Raman Spectroscopic Study of Phase Coexistence in Binary Phospholipid Bilayers.

Binary phospholipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by Raman spectroscopy and differential scanning calorimetry (DSC). We examined features in Raman scattering spectra that are sensitive to the lipid phase and, therefore, could indicate the phase coexistence. It was found that the low-frequency half-width of half-maximum (LHWHM) of the 2850 cm-1 Raman line, corresponding to the symmetric CH2 stretching vibrations, unequivocally reveals the coexisting phospholipids in ordered and disordered conformational states, which correspond to ordered and disordered phases coexistence, in the DPPC mole concentration range from 0.4 to 0.9. The phase coexistence in this concentration range was supported by the particular concentration behavior of the ratio between the intensities of the 2880 cm-1 antisymmetric CH2 vibration line and the 2850 cm-1 symmetric one. It was also shown that the spectral shape of the 1300 cm-1 Raman line, corresponding to the CH2 twisting vibrations, is a good indicator for the phase state and phase coexistence in the phospholipid bilayers. Comparison with the DSC curves confirmed that in the DPPC mole concentration range from 0.4 to 0.9, the two phase transition peaks are observed in DSC curve, those positions are independent of the DPPC concentration. The outcome of the study is the robust label-free contactless approach for the detection of the lipid phase separation, which can be realized with the micrometer resolution.

Nanocage formation and structural anomalies in imidazolium ionic liquid glasses governed by alkyl chains of cations.

Intriguing nanostructuring anomalies have been recently observed in imidazolium ionic liquids (ILs) near their glass transition points, where local density around a nanocaged solute progressively grows up with temperature. Herewith, we for the first time demonstrate experimentally and theoretically, that these anomalies are governed by alkyl chains of cations and crucially depend on their length. Electron Paramagnetic Resonance (EPR) spectroscopy on a series of ILs [Cnmim]BF4 (n = 0-12) shows that only the chains with n = 3-10 favor anomaly. Moreover, remarkable even vs. odd n peculiarities were systematically observed. Finally, similar anomaly was for the first time observed for a non-IL glass of dibutyl phthalate, which structurally mimics cations of imidazolium ILs. Therefore, such anomalous density behavior in a glassy state nanocage goes far beyond ILs and proves to be a more general phenomenon, which can be structurally tuned and rationally adjusted for various potential applications in nanoscale materials.