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The use of deuterated compounds is an interesting opportunity to expand the capabilities of Raman spectroscopy to study metabolism in living cells. Different biological objects have different tolerances to different deuterated compounds, and their metabolic chains may differ. Here, we explore the potential of this approach to probe metabolism in early mouse embryos. We investigated the Raman spectra of mouse embryos at different developmental stages cultured with deuterated amino acids, phenylalanine-d8 and leucine-d10, glucose-d7, and D 2 O. Embryos after in vitro culture with 20 % v/v D 2 O demonstrate Raman peak at 2186 cm -1 corresponding to newly synthesized proteins. Deuterated amino acids can slow down the development rate in 4-8 cell stage embryos, and deuterated glucose can be used at 2 mM concentration. For blastocyst, it was possible to achieve 75 % fraction of deuterated phenylalanine, when cultured with glucose, the maximal intensity ratio between CD and CH bands was 13.7 %. To demonstrate the capabilities of Raman spectroscopy reinforced by deuterium labeling, we investigated the short-term effect of cryopreservation and revealed that cryopreservation decreases the amount of saccharides in embryos and does not affect the activity of protein de novo synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

There is the rapid growth in application of Brillouin scattering spectroscopy to biomedical objects in order to characterize their mechanoelastic properties in this way. However, the possibilities and limitations of the method when applied to tissues have not yet been clarified. Here, applicability of Brillouin spectroscopy for testing the elastic response of medically relevant tissues of bovine jugular vein and pericardium was considered. Parameters of the Brillouin peak were studied for samples untreated, diepoxide-fixed, and preserved after treatment in alcohol solutions. It was found that diepoxide cross-linking resulted to a slight tendency to increase the Brillouin position for hydrated tissues. The variations in the position and width of the Brillouin peaks, associated with local fluctuations in water concentration, were reduced after diepoxide treatment in the case of the pericardium, but not in the case of the vein wall. To obtain more information about the elastic response of the protein scaffold without the participation of water, dried samples were also studied. Brillouin spectra of the dried pericardium and vein wall revealed a significant increase in the Brillouin peak position (elastic modulus) after conservation in alcohol. In the case of the vein wall, this effect was found for both collagen and elastin-related peaks, which were identified in the Brillouin spectrum. This result corresponds to a denser packing of fibrous proteins after preservation in alcohol solutions. The ability of Brillouin spectroscopy to independently characterize the effect of treatment on the instantaneous elastic modulus of various tissue components is also attractive for its application in the development of new materials for bioimplants. A comparison of the Brillouin longitudinal and Young's elastic moduli determined for the hydrated samples of the vein and pericardium showed that there is no clear correspondence between these material parameters. The usefulness of using both experimental methods to obtain new information about the elastic response of the material is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Advances in cryobiology techniques commonly target either the cooling or the warming cycle, while little thought has been given to ≪repair≫ protocols applicable during cold storage. In particular, crystallization is the dominant threat to cryopreserved samples but proceeds from small nuclei that are innocuous if further growth is forestalled. To this end, we propose a laser editing technique that locally heats individual crystals above their melting point by a focused nanosecond pulse, followed by amorphization during rapid resolidification. As a reference, we first apply the approach to ice crystals in cryoprotected solution and use Raman confocal mapping to study the deactivation of crystalline order. Then, we examine dimethyl sulfoxide trihydrate crystals that can germinate at low temperatures in maximally freeze concentrated regions, as commonly produced by equilibrium cooling protocols. We show how to uniquely identify this phase from Raman spectra and evidence retarded growth of laser-edited crystals during warming., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

BSTS epitaxial thin film topological insulators were grown using the MBE technique on two different types of substrates i.e. , Si (111) and SiC/graphene with Bi 0.7 Sb 1.6 Te 1.8 Se 0.9 and Bi 0.9 Sb 1.5 Te 1.8 Se 1.1 , respectively. The crystallographic properties of BSTS films were investigated via X-ray diffraction, which showed the strongest reflections from the (0 0 l ) facets corresponding to the rhombohedral phase. Superior epitaxial growth, homogeneous thickness, smooth surfaces, and larger unit cell parameters were observed for the films grown on the Si substrate. Polarization dependent Raman spectroscopy showed a weak appearance of the A g mode in cross--polarized geometry. In contrast, a strong E g mode was observed in both parallel and cross-polarized geometries which correspond to the rhombohedral crystal symmetry of BSTS films. A redshift of A g and E g modes was observed in the Raman spectra of BSTS films grown on the Si substrate, compared to those on SiC/graphene, which was directly associated with the unit cell parameter and composition of the films. Raman spectra showed four fundamental modes with asymmetric line shape, and deconvolution of the peaks resulted in additional modes in both the BSTS thin films. The sum of relative ratios of linewidths of fundamental modes (A g and E g ) of BSTS films grown on Si substrate was lower, indicating a more ordered structure with lower contribution of defects as compared to BSTS film grown on SiC/graphene substrate.

Under physiological conditions, the membranes and lipid droplets of germ cells are in a conformationally disordered phase. Typically, during cooling, lipids undergo the transition to ordered phases and, upon heating, melt into a disordered phase. In this communication, we report the lipid phase transition in lipid droplets observed in porcine oocytes. Upon cooling, a sharp lipid phase transition from conformationally disordered to ordered state was detected within the temperature range between 20 and 15 °C. Subsequent heating to 45 °C does not return lipids to their original phase state. To the best of our knowledge, this is the first observation of an irreversible phase transition in lipid droplets of biological cells with native lipid composition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Raman spectroscopy of cells cultured in a deuterated substrate is a promising approach to the characterization of mass transfer and enzymatic reactions in living cells. Here, we studied the potential of this approach using the example of yeast cells cultured under aerobic and anaerobic conditions. In our experiments, unadapted to D 2 O Saccharomyces cerevisiae were cultured in a medium with different concentrations of deuterium oxide and deuterated glucose. It has been shown that the addition of even 10% heavy water leads to a general decrease in the amount of lipids in cells. In the Raman spectra of cells cultured at high concentrations of D 2 O, additional peaks are found, which are associated with the deuteration of entire chemical groups. We observed a similar effect in the ethanol synthesized by yeast fermentation, the deuteration of which also depends on the concentration of D 2 O. The results on the characterization of cell deuteration turned out to be in qualitative agreement with the known estimate that aerobic metabolism is 15 times more active than ethanol fermentation. The results of our work determine new limitations and prospects for further application and development of the Raman method of spectroscopy of deuterium tags., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Here, we examined the gigahertz sound velocities of hydrated multibilayers of saturated (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) and unsaturated (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) phospholipids by Brillouin spectroscopy. Out-of-plane and in-plane (lateral) phonons were studied independently of each other. Similar strong temperature dependences of the sound velocities were found for phonons of both types. The sound velocities in the low-temperature limit were two-fold higher than that at physiological temperatures; a significant part of the changes in sound velocity occurs in the solid-like gel phase. The factors that may be involved in the peculiar behavior of sound velocity include changes in the chain conformational state, relaxation susceptibility, changes in the elastic modulus at infinite frequencies, and lateral packing of molecules., Competing Interests: Declaration of Competing Interest Confirm that there is no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Conformational states of phospholipid chains in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC d62 ), and cholesterol (Chol) were studied by Raman spectroscopy. Parameters of Raman peaks sensitive to conformational order have been used to determine chain order for mixtures over a wide range of compositions. A ternary diagram of fractions of phospholipid chains in conformationally ordered and disordered states has been constructed. It was found that the addition of POPC and cholesterol increases the fraction of DPPC chains in disordered conformations. The so-called liquid-ordered phase includes DPPC molecules in both ordered and disordered states in comparable proportions. It was found that POPC chains are partially ordered in mixtures with DPPC and cholesterol, in contrast to the case of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This maybe the underlying reason why ternary mixtures with POPC have different miscibility behavior compared to DOPC., Competing Interests: Declaration of Competing Interest declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., (© 2023 Royal Microscopical Society.)

Carbon nanotubes (CNTs) are used in applications ranging from electrical engineering to medical device manufacturing. It is well known that the addition of nanotubes can influence the mechanical properties of various industrial materials, including plastics. Electrospinning is a popular method for fabricating nanomaterials, widely suggested for polymer scaffold manufacturing. In this study, we aimed to describe the influence of single-walled carbon nanotube (SWCNT) suspensions on polymeric poured films and electrospun scaffolds and to investigate their structural and mechanical properties obtained from various compositions. To obtain films and electrospun scaffolds of 8 mm diameter, we used poly-ε-caprolactone (PCL) and poly(cyclohexene carbonate) (PCHC) solutions containing several mass fractions of SWCNT. The samples were characterized using tensile tests, atomic force and scanning electronic microscopy (AFM and SEM). All the studied SWCNT concentrations were shown to decrease the extensibility and strength of electrospun scaffolds, so SWCNT use was considered unsuitable for this technique. The 0.01% mass fraction of SWCNT in PCL films increased the polymer strength, while fractions of 0.03% and more significantly decreased the polymer strength and extensibility compared to the undoped polymer. The PHCH polymeric films showed a similar behavior with an extremum at 0.02% concentration for strength at break.

The ability of salts to change the macroscopic viscosity of their aqueous solutions is described by the Jones-Dole equation with B-coefficient for the linear concentration term. The sign and value of this coefficient are often considered as a measure of the salt's structure-making/breaking ability, while the validity of this assignment is still under discussion. Here, by applying Raman and Brillouin scattering spectroscopy to various salts from the Hofmeister series, we studied a possible relation between macroscopic Jones-Dole's B-coefficient and the microscopic dynamic response. Raman spectroscopy provides information about molecular vibrations and Brillouin spectroscopy about acoustic phonons with wavelengths of hundreds of nanometers. It has been found that Jones-Dole's B-coefficient correlates linearly with the coefficients, describing the concentration dependences of the average OH stretching frequency, real and imaginary parts of gigahertz elastic modulus. These relationships have been interpreted to mean that the OH stretching frequency is a measure of the ion-induced changes in the water network that cause changes in both viscosity and gigahertz relaxation. Depolarized inelastic light scattering revealed that the addition of structure-making ions not only changes the frequency of the relaxation peak but also increases the low-frequency part of the relaxation susceptibility. It was shown that the ion-induced increase in the gigahertz elastic modulus can be described by changes in the relaxational susceptibility without a noticeable change in the instantaneous elastic modulus. The isotropic Raman contribution associated with the tetrahedral-like environment of H 2 O molecule does not correlate with Jones-Dole's B-coefficient, suggesting a minor influence of these tetrahedral-like configurations on viscosity.

Titanium oxide (TiO 2 ) and oxynitride (N-TiO 2 ) coatings can increase nitinol stents' cytocompatibility with endothelial cells. Methods of TiO 2 and N-TiO 2 sputtering and cytocompatibility assessments vary significantly among different research groups, making it difficult to compare results. The aim of this work was to develop an integral cytocompatibility index (ICI) and a decision tree algorithm (DTA) using the "EA.hy926 cell/TiO 2 or N-TiO 2 coating" model and to determine the optimal cytocompatible coating. Magnetron sputtering was performed in a reaction gas medium with various N 2 :O 2 ratios and bias voltages. The samples' morphology was studied by scanning electron microscopy (SEM) and Raman spectroscopy. The cytocompatibility of the coatings was evaluated in terms of their cytotoxicity, adhesion, viability, and NO production. The ICI and DTA were developed to assess the cytocompatibility of the samples. Both algorithms demonstrated the best cytocompatibility for the sample sputtered at U bias = 0 V and a gas ratio of N 2 :O 2 = 2:1, in which the rutile phase dominated. The DTA provided more detailed information about the cytocompatibility, which depended on the sputtering mode, surface morphology, and crystalline phase. The proposed mathematical models relate the cytocompatibility and the studied physical characteristics.

Cryopreservation of preimplantation embryos is a widely used technique, but this procedure might impact the subsequent embryo development. The effect of slow freezing and vitrification on the lipid metabolism in preimplantation mammalian embryos is not well studied. In this work, we applied Raman spectroscopy of isotopically labeled molecules to address the effects of cryopreservation on fatty acid accumulation in mouse embryos. Embryos after slow freezing or vitrification were cultured for 20 h in a medium supplemented with bovine serum albumin saturated with deuterated stearic acid (dSA). After this period the concentration of dSA estimated from Raman spectra of frozen-thawed and vitrified-warmed embryos at the morula stage was almost twice higher compared to non-cryopreserved morulas. At the same time, frozen-thawed and vitrified-warmed 4-cell embryos did not demonstrate any difference in the level of stearic acid uptake from non-cryopreserved embryos of the same stage. After an additional 24 h culture, cryopreserved and non-cryopreserved embryos demonstrated similar dSA uptake., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Multicomponent lipid bilayers are used as models for searching the origin of spatial heterogeneities in biomembranes called lipid rafts, implying the coexistence of domains of different phases and compositions within the lipid bilayer. The spatial organization of multicomponent lipid bilayers on a scale of a hundred nanometers remains unknown. Brillouin spectroscopy providing information about the acoustic phonons with the wavelength of several hundred nanometers has an unexplored potential for this problem. Here, we applied Brillouin spectroscopy for three binary bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol. The Brillouin experiment for the oriented planar multibilayers was realized for two scattering geometries involving phonons for the lateral and normal directions of the propagation. The DPPC-DOPC mixtures known for the coexistence of the solid-ordered and liquid-disordered phases had bimodal Brillouin peaks, revealing the phase domains with sizes more than a hundred nanometers. Analysis of the Brillouin data for the binary mixtures concluded that the lateral phonons are preferable for testing the lateral homogeneity of the bilayers, while the phonons spreading across the bilayers are sensitive to the layered packing at the mesoscopic scale.

Biomimetic phospholipid mixtures are actively used as models of biological membranes and materials for drug delivery in biomedical tasks. One of the essential properties of membranes formed from complex phospholipid mixtures is the equilibrium coexistence of domains of different phases. Studying the conformational state and chemical content of different phases is of great interest in membrane biophysics. We propose an approach for studying phase coexistence in stacked phospholipid bilayers using Raman mapping. For this purpose, phospholipid multilayer films were formed in which the domains of the same phase were self-aligned in stacks. Raman spectra with a high spectral resolution and signal-to-noise ratio obtained on these samples made it possible to estimate the chemical composition and conformational state of lipids in domains of different phases. For the ternary mixture 1,2-dioleoyl- sn-glycero -3-phosphocholine (DOPC)/1,2-dipalmitoyl-d62- sn-glycero -3-phosphocholine (DPPC-d62)/cholesterol (Chol) used in our demonstration, the phase diagram was studied and the effect of hydration on lipid phase separation was revealed. For the hydrated films, the obtained phase diagram is in qualitative agreement with the previous data obtained using 2 H NMR. In dry films, phase separation is observed for all investigated compositions, with a tendency to form a phase with a high fraction of DPPC-d62. The use of multilayer phospholipid films makes it possible to release the potential of Raman microspectroscopy to study the phase diagrams of phospholipid mixtures under various experimental conditions.

Single-walled carbon nanotubes (SWCNTs) are a perfect host for the formation of one-dimensional phosphorus structures and to obtain hybrid materials with a large P-C ratio. This work presents a procedure for high-yield phosphorus filling of commercial Tuball SWCNTs and efficient removal of phosphorus deposits from the external nanotube surface. We probed white and red phosphorus as precursors, varied the synthesis temperature and the ampoule shape, and tested three solvents for sample purification. High-resolution transmission electron microscopy and Raman spectroscopy indicated crystallization of interior phosphorus in a form resembling fibrous red phosphorus. An aqueous sodium hydroxide solution allowed removing the majority of external phosphorus particles. Thermogravimetric analysis of the product determined ∼23 wt % (∼10 atom %) of phosphorus, and the X-ray photoelectron spectroscopy (XPS) data showed that ca. 80% of it is in the form of elemental phosphorus. Externally purified SWCNTs filled with phosphorus were used to study the interaction between the components. Raman spectroscopy and core-level XPS revealed p-type SWCNT doping. Valence-band XPS data and density functional theory calculations confirmed the transfer of the SWCNT electron density to the encapsulated phosphorus.

Raman spectra of aqueous suspensions of vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC d62 ), and cholesterol (Chol) were studied at room temperature to determine the conformational states of the phospholipid hydrocarbon chains. Deuteration of DPPC d62 allowed us to characterize the conformational states of DOPC and DPPC d62 independently. The parameters of Raman peaks, which are sensitive to the conformational order, were studied in a wide range of compositions. It was found that the DOPC molecules are conformationally disordered for all compositions. The conformational state of the DPPC d62 molecules changes with composition. Their conformational state is influenced by cholesterol-induced partial disordering and DOPC solvation, transforming the DPPC molecules into the disordered state. The conformational state diagram from the Raman experiment was compared with outcomes from the differential scanning calorimetry (DSC) experiment. The Raman spectra also revealed that the DPPC molecules coexist in the disordered and all-trans ordered states for the DOPC/DPPC d62 /Chol mixtures except for the pure liquid-disordered phase., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The use of deuterocarbons is an effective method in the Raman spectroscopy of multicomponent lipid materials and biological samples. Here, Raman spectra of hydrated multilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), its deuterated analog 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC d62 ), and DPPC-DPPC d62 mixtures were studied in a wide temperature range to specify the Raman indicators of conformational and lateral orders. The temperature dependence of the 985 cm -1 line in the deuterated phospholipid unequivocally indicates that this line corresponds to the CC stretching vibrations of deuterated hydrocarbon chains in the all-trans conformation. It was also concluded that the ratio of Raman intensities at the maximum of the peak of the symmetric CD 2 stretching and at a maximum near 2168 cm -1 reflects the conformational order of the hydrocarbon chain and can be used for an evaluation of the fraction of the all-trans sequences. The frequency of the symmetric CD 2 stretching peak is sensitive to the phase state (gel or fluid) but has a low sensitivity to the partial conformational disordering within the gel phase. The Raman study of DPPC-DPPC d62 mixtures reveals that the lateral order contributes to the ratio of intensities of the antisymmetric and symmetric CH 2 stretching peaks as a prefactor enhancing the effect of conformational ordering., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cryopreservation of oocytes has already been used to preserve genetic resources, but this technology faces limitations when applied to the species whose oocytes contain large amounts of cytoplasmic lipid droplets. Although cryoinjuries in such oocytes are usually associated with the lipid phase transition in lipid droplets, this phenomenon is still poorly understood. We applied Raman spectroscopy of deuterium-labeled lipids to investigate the freezing of lipid droplets inside cat oocytes. Lipid phase separation was detected in oocytes cryopreserved by slow-freezing protocol. For oocytes supplemented with stearic acid, we found that saturated lipids form the ordered phase being distributed at the periphery of lipid droplets. When an oocyte is warmed to physiological temperatures after cooling, a fraction of saturated lipids may remain in the ordered conformational state. The fractions of monounsaturated and polyunsaturated lipids redistribute to the core of lipid droplets. Monounsaturated lipids undergo the transition to the ordered conformational state below -10°C. Using deuterated fatty acids with a different number of double bonds, we reveal how different lipid fractions are involved in the lipid phase transition of a cytoplasmic lipid droplet and how they can affect cell survival. Raman spectroscopy of deuterated lipids has proven to be a promising tool for studying the lipid phase transitions and lipid redistributions inside single organelles within living cells., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Low-frequency Raman spectroscopy was used to study the dynamic response on a nanometer scale of aqueous suspensions of two-component lipid vesicles. Binary mixtures of saturated phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) and cholesterol are interesting for possible coexistence of solidlike and liquid-ordered phases, while the phase coexistence was not reported for unsaturated phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and cholesterol mixtures. The DOPC-DPPC mixtures represent the well-documented case of coexisting domains of solidlike and liquid-disordered phases. These three series of lipid mixtures are studied here. A broad peak with the maximum in the range of 30-50cm^{-1} and a narrow peak near 10cm^{-1} are observed in the Raman susceptibility of the binary mixtures and attributed to the acousticlike vibrational density of states and layer modes, respectively. Parameters of the broad and narrow peaks are sensitive to lateral and conformational hydrocarbon chain ordering. It was also demonstrated that the low-frequency Raman susceptibility of multicomponent lipid bilayers allows one to determine the phase state of lipid bilayers and distinguish the homogeneous distribution of molecular complexes from coexisting domains with sizes above several nanometers. Thus, the low-frequency Raman spectroscopy provides unique information in studying phase coexistence in lipid bilayers.

The aqueous suspension of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with different hydration levels α (water-to-lipid mass ratio) have been studied by Brillouin spectroscopy in the temperature range from -190 °C to 70 °C. The samples with different hydration levels demonstrate similar temperature behavior of their sound velocity in the temperature range from -190 °C to -25 °C. There is a strong correlation between the hydration level of the sample and the character of the sound velocity temperature dependence at higher temperatures. Nevertheless, all hydrated samples demonstrate a jump in the sound velocity at the gel-fluid phase transition temperature. The amplitude of this jump depends on the hydration level α of the sample. It has a maximum value in the sample with minimal α necessary for the phospholipid membrane's full hydration. To evaluate the sound velocity in the phospholipid membrane, we applied the two-component model to analyze the experimental data obtained in the sample with α = 0.25 (close to the minimal necessary value for the full DPPC membrane hydration). It was found that for temperatures higher than 0 °C, the two-component model works well if we consider that sound velocity in water between vesicle layers is approximately a factor of two higher than in bulk water., (© 2021 IOP Publishing Ltd.)

We present a comprehensive theoretical and experimental Raman spectroscopic comparative study of bulk Phosphorus allotropes (white, black, Hittorf's, fibrous) and their monolayer equivalents, demonstrating that the application of the Placzek approximation to density functional theory calculated frequencies allows reliable and accurate reproduction of the bulk spectra at a relatively low computational cost. As well as accurate frequencies, peak intensities are also reproduced with reasonable accuracy. Having established the viability of the method we apply it to other less well characterised phosphorus forms such as isolated P4 cages and the planar blue-phosphorus phase. There are several speculative structural models in the literature for amorphous red phosphorus, and we predict Raman spectra for several of these. Via comparison with experiment this allows us to eliminate many of them such as the P2P2-zigzag chain and connected P4 models. The combination of Density functional theory (DFT) modelling, Placzek approximation for intensities with experimental Raman spectroscopy is demonstrated as a powerful combination for accurate characterisation of phosphorus species.

The phospholipid/cholesterol binary model systems are an example of simple models whose structure has caused controversy and genuine interest over many decades. The cornerstone underlying the description of such models is the answer to the question of whether these membranes are separated into coexisting phases or domains. Here, we apply label-free Raman spectroscopy and differential scanning calorimetry (DSC) to verify the phase coexistence in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol binary model. Raman spectra demonstrate the peculiarity at 30% molar fraction of cholesterol. Above this concentration, Raman data demonstrate similar characteristics at T = 291, 298, 303 K. At lower molar fractions, at 303 K, we found the agreement of Raman spectra with the predictions of the lever rule of cholesterol. Taken together, low cooperativity of the transition at 30 mol% and the fulfillment of the lever rule suggest the existence of nanoclusters composed of approximately 4 DMPC and 2 cholesterol molecules. At 298 K, the compliance of the lever rule was found in the range from 0 to 20 mol% of cholesterol. At 291 K, the addition of 5% cholesterol leads to the abrupt change of Raman spectra parameters and their continuous evolution with the further increase of cholesterol molar fraction. It seems that cholesterol plays a twofold role in binary mixtures; it reduces the intermolecular cooperativity and forms clusters whose size and DMPC-to-cholesterol ratio depend on cholesterol concentration and temperature., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report on the reorientation dynamics of small spin probe 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) in cis -1,4-poly(isoprene) ( cis -1,4-PIP10k) from electron spin resonance (ESR) and the free volume of cis -1,4-PIP10k from positron annihilation lifetime spectroscopy (PALS) in relation to the high-frequency relaxations of cis -1,4-PIP10k using light scattering (LS) as well as to the slow and fast processes from broadband dielectric spectroscopy (BDS) and neutron scattering (NS). The hyperfine coupling constant, 2 A zz '( T ), and the correlation times, τ c ( T ), of cis -1,4-PIP10k/TEMPO system as a function of temperature exhibit several regions of the distinct spin probe TEMPO dynamics over a wide temperature range from 100 K up to 350 K. The characteristic ESR temperatures of changes in the spin probe dynamics in cis -1,4-PIP10k/TEMPO system are closely related to the characteristic PALS ones reflecting changes in the free volume expansion from PALS measurement. Finally, the time scales of the slow and fast dynamics of TEMPO in cis -1,4-PIP10k are compared with all of the six known slow and fast relaxation modes from BDS, LS and NS techniques with the aim to discuss the controlling factors of the spin probe reorientation mobility in polymer, oligomer and small molecular organic glass-formers.

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 CH 2 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 CH 2 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 CH 2 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.

Fatty acid uptake and accumulation in lipid droplets are essential processes of lipid metabolism. Oocyte in vitro culture in media enriched with fatty acid is used to modify the lipid content and composition, aiming to study the consequences of obesity and enhance cell cryotolerance. We applied Raman spectroscopy and deuterium labeling approach to quantify stearic acid uptake and investigate its incorporation within oocytes. Our data suggest that deuterium labeling does not affect oocyte maturation rates. The efficiency of deuterated stearic acid (dSA) uptake was shown to decrease with the increase of its concentration in culture medium and the duration of in vitro culture. The molar ratio between dSA and bovine serum albumin has no significant effect on the dSA uptake for 200 μM but modifies concentration dependence of the lipid uptake. dSA accumulates in all the lipid droplets inside oocytes. Different lipid droplets within the same oocyte exhibit different concentrations of dSA. The scatter in the dSA concentration in lipid droplets decreases with the culture time. Using dSA as an example, we provide a comprehensive description of how fatty acid concentration, its molar ratio versus bovine serum albumin, and culture time affect the uptake of the fatty acids in oocytes. Raman microspectroscopy of deuterium-labeled fatty acids is a nondestructive tool providing information about fatty acid uptake and heterogeneity of their accumulation between lipid droplets within the single oocyte., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Suspensions of multilamellar vesicles of 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) and hydrated and dehydrated aligned multilamellar samples of DMPC were studied by Brillouin spectroscopy in the temperature range from 90 to 333 K. The sound velocity of the longitudinal acoustic wave was evaluated from the Brillouin spectra. It was found that phase transition, hydration state, and planar or vesicular form of bilayers affect the gigahertz sound velocity. Usually, the temperature dependence of the sound velocity is weak in solid substances. Amazingly, the sound velocity of hydrated DMPC samples showed significant temperature-induced changes of up to 1.8 times, even within the solid-like gel phase. We explained this effect by temperature-induced excitations of the disordered conformational states of the hydrocarbon chains as well as anharmonic effects. In addition, the relevance of the gigahertz sound velocity to the description of subterahertz Raman features was demonstrated.

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 [C n mim]BF 4 (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.

Small sugars are known to stabilize biological membranes under extreme conditions of freezing and desiccation. The proposed mechanisms of stabilization suggest membrane-sugar interactions to be either attractive or repulsive. To obtain new insight into the problem, we use a recently developed low-frequency Raman scattering approach which allows detecting membrane mechanical vibrations. For model membranes of palmitoyl-oleoyl-glycero-phosphocholine (POPC) hydrated in aqueous sucrose and trehalose solutions, we studied the Raman peak between 12 and 15 cm -1 that is attributed to an eigenmode of the normal mechanical vibrations of a lipid monolayer. For both sugars, similar results were obtained. With an increase in sugar concentration in solution, the frequency position of the peak was found to decrease by ∼13% which was interpreted as a consequence of the membrane thickening due sugar monolayer adsorption on the membrane surface. The concentration dependence of the peak frequency position was satisfactorily described by a Langmuir monolayer adsorption model. It is concluded that, at small sugar concentrations (less than 0.2 M), the membrane-sugar interactions are attractive, while at higher concentrations (more than 0.4 M) the attraction disappears. The data obtained show that one sugar molecule on the surface interacts with approximately 3-4 polar lipid heads.

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4-8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (-2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation., (© 2020 Blackwell Verlag GmbH.)

The phase relations in the subsolidus region of the Tl 2 MoO 4 -Bi 2 (MoO 4 ) 3 -Hf(MoO 4 ) 2 system were studied with the "intersecting cuts" method. The formation of the novel ternary molybdate Tl 5 BiHf(MoO 4 ) 6 is found in this ternary system. The compound has a phase transition at T pt = 731 K (Δ H = -3.15 J/g) and melts at T m = 871 K (Δ H = -41.71 J/g), as determined by a thermal analysis. Tl 5 BiHf(MoO 4 ) 6 single crystals were obtained by the spontaneous nucleation method. The crystal structure of Tl 5 BiHf(MoO 4 ) 6 was revealed by structure analysis methods. This molybdate crystallizes in the trigonal space group R 3̅ c with the unit cell parameters a = 10.6801(4) Å, c = 38.5518(14) Å, V = 3808.3(2) Å 3 , and Z = 6. The vibrational characteristics of Tl 5 BiHf(MoO 4 ) 6 were determined by Raman spectroscopy. The Tl 5 BiHf(MoO 4 ) 6 conductivity was measured at frequencies of 0.1, 1.0, and 10 kHz in the temperature range of 293-773 K; in this temperature range, the conductivity level was 10 -12 -10 -7 S/cm.

Raman spectra of hydrated bilayers of 1,2-dilauroyl-sn-glycero-3-phosphocholine (12:0 PC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0 PC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC) were studied in a wide temperature range to characterize the temperature-dependent hydrocarbon chain disorder of the saturated phosphatidylcholines. Temperature dependencies of the Raman line intensities were analyzed for the antisymmetric CH 2 stretching and CC stretching vibrations, which are sensitive to the lipid order. It was found that chain disordering processes occur significantly below the gel-fluid transition. The chain conformational order characterized by the CC stretching line intensity can be well described by a model involving the excitation of the ordered conformational state to the kinked and highly disordered, fluid-like state. A relation between the excitation energy to the disordered state and the enthalpy of the gel-fluid transition is discussed, including also data for other phosphatidylcholines studied before. Temperature behavior of the antisymmetric CH 2 stretching line indicates that non-conformational degrees of freedom are released above ∼ 200 K. Experimental findings concerning the hydrocarbon chain length dependence of the Raman polarizability of antisymmetric CH 2 stretching vibrations and a low-temperature solid-solid phase transition, identified in 12:0 PC at heating, are also discussed in the work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although lipid droplets are believed to play an important role in cryopreservation of mammalian embryos and oocytes, the effect of low temperatures on lipid droplets and related mechanisms of cryodamage are still obscure. Here, we provide Raman spectroscopy evidence of lipid separation inside the lipid droplets in domestic cat oocytes during slow freezing. It was shown that at -25 °C lipids coexist in two separated phase states inside lipid droplets. The scale of detected domains was a few micrometers size. We also found that under certain conditions these areas have a specific spatial distribution. Lipids with high melting temperatures are distributed near the surface of lipid droplets while fusible lipids are located deep inside. Raman spectroscopy was found to be a prospective approach to study inhomogeneity of lipid phase transition in cells and to reveal effects of this inhomogeneity on cryopreservation of biological cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recently it was shown [V. N. Novikov and A. P. Sokolov, Phys. Rev. E 92, 062304 (2015)10.1103/PhysRevE.92.062304] that the second derivative with respect to inverse temperature of the structural relaxation time in some supercooled molecular liquids has a sharp maximum. It marks the point at which the apparent activation energy begins to saturate with decreasing temperature. The elastic model of glass-forming liquids expresses the temperature dependence of the structural relaxation time through that of the shear modulus. In this paper, we test whether this model is able to predict the maximum of the second derivative. We confirm its presence in the elastic model by analyzing the temperature dependence of the Brillouin light scattering in salol. This is a very subtle feature of the temperature dependence, which is greatly enhanced when taking derivatives. Its presence in the Brillouin data provides strong support to the elastic model of glass-forming liquids.

The orientation of lipid molecules is an essential characteristic of supported phospholipid layers, synthetic lipid structures, and biological specimens. Here, we perform Raman spectroscopy to analyze the orientation order in lipid structures. For this purpose, we studied dry oriented planar DMPC samples and multilamellar DPPC vesicles in water using Raman mapping. Principal component analysis (PCA) was applied to extract the information about the orientational order of lipid molecules. Using PCA, we revealed the features observed in the phospholipid spectra that are sensitive to hydrocarbon chain orientation relative to the polarization of laser radiation. These spectral features include Raman peaks corresponding to stretching C-C, twisting CH2, rocking and stretching CH3 modes. We suggest to use them as markers of hydrocarbon chain orientation along with light polarization. The proposed Raman analysis can be used to study samples with different levels of hydration.

A lipid bilayer containing a ternary mixture of low- and high-melting lipids and cholesterol (Chol) can give rise to domain formation, referred to as lipid rafts. Low-frequency Raman spectroscopy at reduced temperatures allows detection of normal membrane mechanical vibrations. In this work, Raman spectra were obtained in the spectral range between 5 and 90 cm -1 for bilayers prepared from dioleoyl- glycero -phosphocholine (DOPC), dipalmitoyl- glycero -phosphocholine (DPPC) and Chol. A narrow peak detected between 13 and 16 cm -1 was attributed to the vibrational eigenmode of a lipid monolayer (a leaflet). For the equimolar DOPC/DPPC ratio, the Chol concentration dependence for the peak position, width and amplitude may be divided into three distinct ranges: below 9 mol%, the intermediate range between 9 mol% and 38 mol%, and above 38 mol%. In the intermediate range the peak position attains its minimum, and the peak width drops approximately by a factor of two as compared with the Chol-free bilayers. Meanwhile, this range is known for raft formation in a fluid state. The obtained results may be interpreted as evidence that bilayer structures in the raft-containing fluid state may be frozen at low temperatures. The drop of peak width indicates that at the spatial scale of the experiment (∼2.5 nm) the intermolecular bilayer structure with raft formation becomes more homogeneous and more cohesive., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Embryos cryopreservation is a widely used technology for genetic resources storage. Cryopreservation suppresses cell respiration, but very little is known about the changes that occur with mitochondria at low temperatures. We used Raman spectroscopy to investigate photoresponse and redox state of cytochromes in the respiratory electron transport chain (ETC) in early mouse embryos during cooling. Redox state of cytochromes was probed by the intensity of cytochrome resonance Raman lines. Photoinduced reactions of cytochromes were used to study the changes in the rates of redox reactions. It is found that the rate of cytochrome photoresponse detected by Raman spectra abruptly changes when embryos are cooled below -50 °C. Raman mapping revealed that the average intensity of cytochrome Raman peaks at -65 °C is higher than at -40 °C. Cytochrome b reduction was found in embryos frozen below -50 °C when irradiated with 532 nm laser radiation. These effects were observed for cells frozen in aqueous solutions of two different cryoprotectants: glycerol and propylene glycol. Raman spectroscopy of cytochromes reveals the abrupt changes in the ETC work of frozen mouse embryos at temperatures below -50 °C. We suggest that similar phenomena can be found in various cell types., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The measurement of intracellular viscoelastic properties by Brillouin scattering is a rapidly developing field in biophysics and medicine. Here, the Brillouin spectroscopy is applied for a number of aqueous solutions of biorelevant molecules to reveal relations between the Brillouin line parameters (frequency and width) and viscosity or solute concentration. It is found that for the majority of the studied biorelevant molecules the solute concentration governs the Brillouin frequency in a universal manner. On the other hand, the relations between the macroscopic viscosity and Brillouin peak parameters are different for different solutes. We conclude that for biological fluids the viscosity evaluation from Brillouin data needs prior knowledge about the chemical composition. This result challenges the fidelity of the indirect experimental determinations of the cellular viscosity, when small molecule solutions are used for the calibration.

Raman spectra in the low-frequency spectral range-between 5 and 90cm^{-1}-were studied for multilamellar bilayers prepared with cholesterol (Chol) and phospholipids of three different types: doubly unsaturated lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), monounsaturated lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and fully saturated lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The narrow peak seen below 250 K and positioned between 9 and 18cm^{-1}-depending on the system and temperature-was attributed to the vibrational eigenmode of a lipid monolayer. For the DOPC-Chol bilayer, the peak position and the peak width were found to monotonically increase and decrease, respectively, with the Chol concentration. For POPC-Chol and DMPC-Chol bilayers, these parameters revealed nonmonotonic concentration dependences, with an apparent minimum at the intermediate Chol content. The peak intensity was ascribed to interleaflet coupling. As in the literature, a coexistence of liquid-ordered and solid-ordered domains was suggested for the DMPC-Chol and POPC-Chol bilayers; the Chol concentration dependences of Raman peak parameters were discussed in line with this suggestion, under the assumption that the different composition of coexisting domains conserves upon cooling. We demonstrated that the obtained Raman data disagree with the suggested domain coexistence if the domain sizes are substantially larger than the lipid layer thickness.

We applied a Raman spectroscopy approach to investigate the effect of a cryoprotectant on the redox state of cytochromes on freezing yeast cells. The redox activity of cytochromes was studied using time-resolved photobleaching of the resonance Raman lines. It is found that ice formation causes a drastic change in the redox state of cytochromes in cells frozen without cryoprotectant, whereas in the presence of glycerol the effects of ice formation are more gradual. The photobleaching rate of cells frozen in glycerol solution shows a gradual slowing with temperature decrease and an abrupt slowdown below - 48 °C. This abrupt decrease was interpreted as originating from changes in protein conformational dynamics. Our findings provide important new insights into the transition from active to inactive cytochrome states as cells undergo freezing in the presence and absence of cryoprotectant.

Unusual physical and chemical properties of ionic liquids (ILs) open up prospects for various applications. We report the first observation of density/rigidity heterogeneities in a series of ILs near the glass transition temperature ( T g ) by means of pulse electron paramagnetic resonance (EPR). Unprecedented suppression of molecular mobility is evidenced near the glass transition, which is assigned to unusual structural rearrangements of ILs on the nanometer scale. Indeed, pulse and continuous wave EPR clearly indicate the occurrence of heterogeneities near T g , which exist in a rather broad temperature range of ∼50 K. The two types of local environments are evidenced, being drastically different by their stiffness. The more rigid one suppresses molecular mobility, whereas the softer one instead promotes diffusive molecular rotation. Such properties of ILs near T g are of general importance; moreover, the observed density/rigidity heterogeneities controlled by temperature might be considered as a new type of tunable reaction nanoenvironment.

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from -10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic β and β' phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Although lipid phase transition is believed to be among the major damaging factors in oocytes and preimplantation embryos cryopreservation, lack of the appropriate experimental methods limits investigation of this phenomenon. Herein, we demonstrate the capabilities of Raman spectroscopy to detect the lipid phase transition within the freezing preimplantation mouse embryos. We exploit the sensibility of antisymmetric CH 2 Raman peak to the phase state of lipids. It is shown that during the freezing of the mouse embryos the lipid phase transition occurs at the temperatures between -7 and 0 °C. Similar temperature dependences of CH 2 mode intensities are found for lipids in the preimplantation embryos and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, implying the similarity in the occupation rules of conformational states. Raman spectroscopy is considered as a method of choice to study the lipid phase transition during preimplantation mammalian embryos freezing and cryopreservation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rayleigh-Brillouin light scattering is studied in liquid water over the range from 249 to 365 K. Experiments are carried out with a high spectral resolution (0.1 GHz), eliminating any contribution of the structural relaxation to the elastic line. The Landau-Placzek ratio is found as the ratio of the Rayleigh and Brillouin intensities. In the whole temperature range, the Landau-Placzek ratio is found to be in good agreement with a prediction of the theory with a pair of independent thermodynamic variables, pressure and entropy. This description is usually used for single-component homogeneous liquids. An excess of the Landau-Placzek ratio above the prediction is expected for inhomogeneous liquids and is observed, for example, in glass-forming liquids below a certain temperature. In contrast to glass-forming liquids, no excess of elastically scattered light increasing at low temperatures is observed for the Landau-Placzek ratio of water. This suggests that the Landau-Placzek ratio of liquid water can be described by a homogeneous structure, and the idea of the water structure consisting of two structural motifs may not be necessary to explain the experimental ratio.

Low-frequency Raman spectra of multilamellar vesicles made either of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) have been studied in a wide temperature range. Below 0^{∘}C two peaks are found at frequencies around 8-9 and 14-17cm^{-1} and attributed to the normal vibrational modes of the phospholipid bilayer, which are determined by the bilayer thickness and stiffness (elastic modulus). The spectral positions of the peaks depend on the temperature and the bilayer composition. It is suggested that the ratio of the intensities of the first and second peaks can serve as a measure of the interleaflet elastic coupling. The addition of cholesterol to the phospholipid bilayer leads to peak shift and broadening, which may be assigned to the composition heterogeneities commonly attributed to the lipid raft formation.

The distribution of cryoprotectant (10% glycerol) and ice along the frozen plastic straw (the most useful container for freezing mammalian semen, oocytes and embryos) was studied by Raman scattering technique. Raman spectroscopy being a contactless, non-invasive tool was applied for the straws filled with the cryoprotectant solution and frozen by controlled rate programs commonly used for mammalian embryos freezing. Analysis of Raman spectra measured at different points along the straw reveals a non-uniform distribution of the cryoprotectant. The ratio between non-crystalline solution and ice was found to be increased by several times at the bottom side of the solution column frozen by the standard freezing program. The increase of the cryoprotectant fraction occurs in the area where embryos or oocytes are normally placed during their freezing. Possible effects of the cooling rate and the ice nucleation temperature on the cryoprotectant fraction at the bottom side of the solution column were considered. Our findings highlight that the ice fraction around cryopreserved embryos or oocytes can differ significantly from the averaged one in the frozen plastic straws., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Raman scattering of five phosphatidylcholines [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (PC24)] was studied in a wide temperature range. These phospholipid bilayers differ in the temperature of the gel-fluid transition Tm (from -57 to +80 °C) and in the number of unsaturated bonds. For all lipids, the temperature dependence of both asymmetrical methylene stretching and C-C stretching bands provides evidence that the disordering processes occur significantly below Tm. Temperature onset of the decrease of Raman intensity of the asymmetrical methylene stretching band is the same for unsaturated lipids (DLPC, POPC, and DOPC), which was interpreted as the importance of packing defects in the bilayers of these phospholipids. The chain conformational order was characterized by Raman intensity of the high-frequency C-C stretching mode. An approach was used where the Raman intensity of this mode serves as a measure of hydrocarbon chains in the ground conformational state. Temperature dependence of the chains in the ground conformational state was well described by a simple model with the ground and two excited states: the kinked state and the highly disordered, fluidlike state.

Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from -173 to +25 °C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼10(15) times slower at liquid nitrogen temperature than at room temperature., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Diamond attracts considerable attention as a versatile and technologically useful material. For many demanding applications, such as recently emerged quantum optics and sensing, it is important to develop new routes for fabrication of diamond containing defects with specific optical, electronic and magnetic properties. Here we report on successful synthesis of diamond from a germanium-carbon system at conditions of 7 GPa and 1,500-1,800 °C. Both spontaneously nucleated diamond crystals and diamond growth layers on seeds were produced in experiments with reaction time up to 60 h. We found that diamonds synthesized in the Ge-C system contain a new optical centre with a ZPL system at 2.059 eV, which is assigned to germanium impurities. Photoluminescence from this centre is dominated by zero-phonon optical transitions even at room temperature. Our results have widened the family of non-metallic elemental catalysts for diamond synthesis and demonstrated the creation of germanium-related optical centres in diamond.