Raman spectroscopy of yeast cells cultured on a deuterated substrate.

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 D2O 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 D2O, 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 D2O. 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.

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.

Integral Algorithms to Evaluate TiO2 and N-TiO2 Thin Films' Cytocompatibility.

Titanium oxide (TiO2) and oxynitride (N-TiO2) coatings can increase nitinol stents' cytocompatibility with endothelial cells. Methods of TiO2 and N-TiO2 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/TiO2 or N-TiO2 coating" model and to determine the optimal cytocompatible coating. Magnetron sputtering was performed in a reaction gas medium with various N2:O2 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 Ubias = 0 V and a gas ratio of N2:O2 = 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.

Coexistence of lipid phases in multilayer phospholipid films probed by Raman mapping.

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 2H 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.

Thermochemical Laser-Induced Periodic Surface Structures Formation by Femtosecond Laser on Hf Thin Films in Air and Vacuum.

Thermochemical laser-induced periodic surface structures (TLIPSS) are a relatively new type of periodic structures formed in the focal area of linear polarized laser radiation by the thermally stimulated reaction of oxidation. The high regularity of the structures and the possibility of forming high-ordered structures over a large area open up possibilities for the practical application for changing the optical and physical properties of materials surface. Since the mechanism of formation of these structures is based on a chemical oxidation reaction, an intriguing question involves the influence of air pressure on the quality of structure formation. This paper presents the results on the TLIPSS formation on a thin hafnium film with fs IR laser radiation at various ambient air pressures from 4 Torr to 760 Torr. Despite the decrease in the oxygen content in the ambient environment by two orders of magnitude, the formation of high-ordered TLIPSS (dispersion in the LIPSS orientation angle δθ < 5°) with a period of ≈700 nm occurs within a wide range of parameters variation (laser power, scanning speed). This behavior of TLIPSS formation is in agreement with experimental data obtained earlier on the study of the kinetics of high-temperature oxidation of hafnium at various oxygen pressures.

Lipid phase transitions in cat oocytes supplemented with deuterated fatty acids.

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.

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.

Effects of slow freezing and vitrification on embryo development in domestic cat.

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.

Deciphering the orientation of lipid molecules by principal component analysis of Raman mapping data.

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.

Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy.

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 ß and ß' 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.

Effect of glycerol on photobleaching of cytochrome Raman lines in frozen yeast cells.

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.

Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy.

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.

Photobleaching of the resonance Raman lines of cytochromes in living yeast cells.

The photobleaching of the resonance cytochrome Raman lines in living Saccharomyces cerevisiae cells was studied. The photobleaching rate versus the irradiation power was described by square function plus a constant in contrast to the linear dependence of the photoinjury rate. This difference distinguishes the cytochrome photooxidation from other processes of the cell photodamage. The square dependence is associated with the reaction involving two photogenerated intermediates while the constant with the dark redox balance rates. This work demonstrates a potential of Raman spectroscopy to characterize the native cytochrome reaction rates and to study the cell photodamage precursors.