Reversal of crystallization in cryoprotected samples by laser editing.

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.

Irreversible lipid phase transition detected in a porcine oocyte at chilling.

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.

Effects of stearic acid on the embryo cryopreservation in mouse.

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

Cryopreservation increases accumulation of exogenous stearic acid in mouse embryos.

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.

Alteration of the lipid phase transition during mouse embryos freezing after in vitro culture with linoleic acid.

Lipids significantly affect embryo cryopreservation in some mammalian species depending on the cell lipidome quantity and composition. One of the ways to study the relationship between lipid content and cryotolerance of cells is to study the effect of lipidome modification on laboratory mice. The objective of this research was to study how in vitro culture of mouse embryos with linoleic acid (LA) will affect lipid phase transition (LPT) during cooling and subsequent embryo development after cryopreservation. Embryos obtained in vivo at the 2-cell stage were cultured with 200 µM LA for 46 h up to the morula/blastocyst stage. Thereafter, one portion of embryos was slowly frozen to reveal the effect of LA on survival after cryopreservation, another portion was used to characterize the lipid composition and to determine the temperature of the LPT onset. Confocal fluorescence microscopy of Nile Red stained embryos showed a significant increase in lipid content of the LA treated group compared to the controls. Raman measurements showed that the onset of LPT in LA treated embryos is lower than in untreated ones: -5 °C vs +2 °C. However, these changes in the LPT onset did not affect the survival rates of embryos after cryopreservation. In summary, in vitro culture with LA changes the biophysical characteristics of embryos' lipidome and is realized in lower LPT onset, but this does not affect embryo survival after cryopreservation.

Deuterated stearic acid uptake and accumulation in lipid droplets of cat oocytes.

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.

Raman spectroscopy evidence of lipid separation in domestic cat oocytes during freezing.

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.

Effect of low temperatures on cytochrome photoresponse in mouse embryos.

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.

Oxide composition and period variation of thermochemical LIPSS on chromium films with different thickness.

In this paper, we present the results of thermochemical LIPSS formation on a chromium film with a thickness in the range of 28-350 nm induced by femtosecond laser radiation (λ = 1026 nm, ν = 200 kHz, τ = 232 fs). The period, height, morphology and chemical composition of TLIPSS as a function of the metal film thickness and focusing configuration are investigated. The growth of TLIPSS period from 678 nm to 950 nm with increasing thickness of the film has been explained by a formation of oxides with different stoichiometry composition. So, the CrO2 oxide prevails in the composition for the case of TLIPSS formed on thin films which have the minimal period, whereas Cr2O3 oxide is dominant in the case of TLIPSS formed on thick chromium films which have the maximal period value. The results obtained are in agreement with numerical modeling of a period defined by the interference between an incident radiation and a scattered one from a single oxide ridge with a different chemical composition.

Raman spectroscopy reveals the lipid phase transition in preimplantation mouse embryos during freezing.

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 CH2 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 CH2 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.

Cryoprotectant redistribution along the frozen straw probed by Raman spectroscopy.

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.

Raman spectroscopy for DNA quantification in cell nucleus.

Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate.

[Embryos and gametes cryopreservation for genetic resources conservation of laboratory animals].

Article reviews the use of embryos and gametes cryopreservation for cryobanking the laboratory animal species. The special emphasis is made on the mechanisms of cryoinjury and cryoprotection during program freezing and vitrification. The species specific cryobanking problems are discussed and the prospects to overcome these problems are outlined.

Probing metabolism in mouse embryos using Raman spectroscopy and deuterium tags.

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 D2O. Embryos after in vitro culture with 20 % v/v D2O 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.

Raman scattering evidence of hydrohalite formation on frozen yeast cells.

We studied yeast cells in physiological solution during freezing by Raman microspectroscopy technique. The purpose was to find out the origin of a sharp peak near ∼3430cm(-1) in Raman spectrum of frozen mammalian cells, observed earlier (J. Dong et al., Biophys. J. 99 (2010) 2453), which presumably could be used as an indicator of intracellar ice appearance. We have shown that this line (actually doublet of 3408 and 3425cm(-1)) corresponds to Raman spectrum of hydrohalite (NaCl⋅2H(2)O), which is formed as the result of the eutectic crystallization of the liquid solution around the cells. We also show that the spatial distribution of hydrohalite in the sample significantly depends on the cooling rate. At lower cooling rate (1°C/min), products of eutectic crystallization form layer on the cell surface which thickness varies for different cells and can reach ∼1µm in thickness. At higher cooling rate (20°C/min), the hydrohalite distribution appears more homogeneous, in the sample, and the eutectic crystallization layer around the cells was estimated to be less than ∼20nm. These experimental results are consistent with scenarios predicted by the two-factor hypothesis for freezing induced cell injury. This work demonstrates a potential of Raman microspectroscopy to study peculiarities of the eutectic crystallization around single cells in vivo with the high spatial resolution.

Raman spectroscopy and DSC assay of the phase coexistence in binary DMPC/cholesterol multilamellar vesicles.

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.

[Effects of a high-fat diet on the lipid profile of oocytes in mice]. / Влияние диеты с повышенным содержанием жира на липидный профиль ооцитов мышей.

There are evidences that obese women exhibit a detrimental oocyte quality. However, it remains unclear how this change is associated with obesity, indirectly - or directly through a change in the content and/or composition of lipids in oocytes. The aim of this work was to study effects of a high-fat diet applied to female donor mice on the amount and qualitative composition of lipids of immature and in vivo matured oocytes. A high-fat diet caused larger body weight in female mice compared with the control ( p < 0.001; 44.77 ± 1.46 and 35.22 ± 1.57, respectively), and increased the blood levels of cholesterol ( p < 0.05; 2.06 ± 0.10 and 1.78 ± 0.10, respectively) and triglycerides ( p < 0.05; 2.13 ± 0.23 and 1.49 ± 0.21, respectively). At the same time, this diet does not affect the level of unsaturation of lipids in immature (0.207 ± 0.004 in the experiment and 0.206 ± 0.002 in the control) and matured oocytes (0.212 ± 0.005 in the experiment and 0.211 ± 0.003 in the control). Total lipid content increased during in vivo maturation of mouse oocytes. The amount of lipids was greater in mature oocytes in the experimental group compared to the control ( p < 0.01; 8.15 ± 0.37 and 5.83 ± 0.14, respectively). An increase in intracellular lipid amount during oocyte maturation was revealed both after a standard diet ( p < 0.05; 4.72 ± 0.48 and 5.83 ± 0.14, respectively) and after a fat-rich diet ( p < 0.001; 3.45 ± 0.62 and 8.15 ± 0.37, respectively). Thus, during in vivo oocyte maturation in mice the content of intracellular lipids enhanced, the high-fat diet aggravated this dynamics of lipid increase during in vivo maturation of oocytes.

Brillouin spectroscopy of biorelevant fluids in relation to viscosity and solute concentration.

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.