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Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast.

ABSTRACT: The glass-phase densities at T = 77?K of aqueous solutions of the common cryoprotective agents (CPAs) methanol, ethanol, 2-propanol, glycerol, 2-methyl-2,4-pentanediol (MPD), ethylene glycol, polyethylene glycol 200 and polypropylene glycol 425 were measured as a function of CPA concentration. Individual drops with volumes as small as ?65?pl were rapidly cooled to achieve the glass phase, and their densities at T = 77?K were determined by cryoflotation. These densities were used to determine the glass-phase electron density of each solution and its volume thermal contraction between room temperature and 77?K. When combined with data for the critical cooling rates required to achieve the glass phase versus CPA concentration, these yield alternative measures of cryoprotectant effectiveness. These reference data will aid in minimizing sample stresses and mechanical damage in cryocrystallography, in cryogenic temperature X-ray imaging and in vitrification-based cryopreservation protocols, and in maximizing electron-density contrast between cryoprotectant solutions and biomolecules in cryogenic temperature small-angle X-ray scattering experiments and cryo-electron microscopy.

SUBMITTER: Tyree TJ

PROVIDER: S-EPMC5930352 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast.

Tyree Timothy J TJ Dan Ritwik R Thorne Robert E RE

Acta crystallographica. Section D, Structural biology 20180427 Pt 5

The glass-phase densities at T = 77 K of aqueous solutions of the common cryoprotective agents (CPAs) methanol, ethanol, 2-propanol, glycerol, 2-methyl-2,4-pentanediol (MPD), ethylene glycol, polyethylene glycol 200 and polypropylene glycol 425 were measured as a function of CPA concentration. Individual drops with volumes as small as ∼65 pl were rapidly cooled to achieve the glass phase, and their densities at T = 77 K were determined by cryoflotation. These densities were used to determine the ...[more]

PMID: 29717718

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Project description:ObjectivePreservation of biological materials at subzero Centigrade temperatures, cryopreservation, is important for the field of tissue engineering and organ transplantation. Our group is studying the use of isochoric (constant volume) systems of aqueous solution for cryopreservation. Previous studies measured the pressure-temperature relations in aqueous isochoric systems in the temperature range from 0°C to - 20°C. The goal of this study is to expand the pressure-temperature measurement beyond the range reported in previous publications.Materials and methodsTo expand the pressure-temperature measurements beyond the previous range, we have developed a new isochoric device capable of withstanding liquid nitrogen temperatures and pressures of up to 413 MPa. The device is instrumented with a pressure transducer than can monitor and record the pressures in the isochoric chamber in real time. Measurements were made in a temperature range from - 5°C to liquid nitrogen temperatures for various solutions of pure water and Me2SO (a chemical additive used for protection of biological materials in a frozen state and for vitrification (glass formation) of biological matter). Undissolved gaseous are is carefully removed from the system.ResultsTemperature-pressure data from - 5°C to liquid nitrogen temperature for pure water and other solutions are presented in this study. Following are examples of some, temperature-pressure values, that were measured in an isochoric system containing pure water: (- 20°C, 187 MPa); (-25°C, 216 MPa); (- 30°C, 242.3 MPa); (-180°C, 124 MPa). The data is consistent with the literature, which reports that the pressure and temperature at the triple point, between ice I, ice III and water is, - 21.993°C and 209.9 MPa, respectively. It was surprising to find that the pressure in the isochoric system increases at temperatures below the triple point and remains high to liquid nitrogen temperatures. Measurements of pressure-temperature relations in solutions of pure water and Me2SO in different concentrations show that, for concentrations in which vitrification is predicted, no increase in pressure was measured during rapid cooling to liquid nitrogen temperatures. However, ice formation either during cooling or warming to and from liquid nitrogen temperatures produced an increase in pressure.ConclusionsThe data obtained in this study can be used to aid in the design of isochoric cryopreservation protocols. The results suggest that the pressure measurement is important in the design of "constant volume" systems and can provide a simple means to gain information on the occurrence of vitrification and devitrification during cryopreservation processes of aqueous solutions in an isochoric system.

Project description:Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitro-gen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å-2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.

Dataset Information

Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast.

Publications

Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast.

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