scholarly journals High-resolution single-particle cryo-EM of samples vitrified in boiling nitrogen

IUCrJ ◽  
2021 ◽  
Vol 8 (6) ◽  
Author(s):  
Tyler Engstrom ◽  
Jonathan A. Clinger ◽  
Katherine A. Spoth ◽  
Oliver B. Clarke ◽  
David S. Closs ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Single Particle ◽  
Pure Water ◽  
Small Samples ◽  
Dominant Factor ◽  
Primary Coolant ◽  
Cooling Rates ◽  
Leidenfrost Effect ◽  
Induced Motion ◽  
Cold Gas

Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitrogen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitrogen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s−1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitrogen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitrogen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitrogen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

scholarly journals Hyperquenching for protein cryocrystallography

2006 ◽  
Vol 39 (6) ◽  
pp. 805-811 ◽  
Author(s):  
Matthew Warkentin ◽  
Viatcheslav Berejnov ◽  
Naji S. Husseini ◽  
Robert E. Thorne
Keyword(s):  
Liquid Nitrogen ◽  
Best Practice ◽  
Protein Crystals ◽  
Small Samples ◽  
Aqueous Mixtures ◽  
X Ray ◽  
Crystallization Conditions ◽  
Water Crystallization ◽  
Gas Layer ◽  
Cold Gas

When samples having volumes characteristic of protein crystals are plunge cooled in liquid nitrogen or propane, most cooling occurs in the cold gas layer above the liquid. By removing this cold gas layer, cooling rates for small samples and modest plunge velocities are increased to 1.5 × 104 K s−1, with increases of a factor of 100 over current best practice possible with 10 µm samples. Glycerol concentrations required to eliminate water crystallization in protein-free aqueous mixtures drop from ∼28%w/vto as low as 6%w/v. These results will allow many crystals to go from crystallization tray to liquid cryogen to X-ray beam without cryoprotectants. By reducing or eliminating the need for cryoprotectants in growth solutions, they may also simplify the search for crystallization conditions and for optimal screens. The results presented here resolve many puzzles, such as why plunge cooling in liquid nitrogen or propane has, until now, not yielded significantly better diffraction quality than gas-stream cooling.

scholarly journals A comparative study of single-particle cryo-EM with liquid-nitrogen and liquid-helium cooling

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 1099-1105 ◽  
Author(s):  
Olivia Pfeil-Gardiner ◽  
Deryck J. Mills ◽  
Janet Vonck ◽  
Werner Kuehlbrandt
Keyword(s):  
Radiation Damage ◽  
Liquid Helium ◽  
Liquid Nitrogen ◽  
Single Particle ◽  
Particle Analysis ◽  
Data Sets ◽  
Image Recording ◽  
Potential Map ◽  
Induced Motion ◽  
Effects Of Radiation

Radiation damage is the most fundamental limitation for achieving high resolution in electron cryo-microscopy (cryo-EM) of biological samples. The effects of radiation damage are reduced by liquid-helium cooling, although the use of liquid helium is more challenging than that of liquid nitrogen. To date, the benefits of liquid-nitrogen and liquid-helium cooling for single-particle cryo-EM have not been compared quantitatively. With recent technical and computational advances in cryo-EM image recording and processing, such a comparison now seems timely. This study aims to evaluate the relative merits of liquid-helium cooling in present-day single-particle analysis, taking advantage of direct electron detectors. Two data sets for recombinant mouse heavy-chain apoferritin cooled with liquid-nitrogen or liquid-helium to 85 or 17 K were collected, processed and compared. No improvement in terms of resolution or Coulomb potential map quality was found for liquid-helium cooling. Interestingly, beam-induced motion was found to be significantly higher with liquid-helium cooling, especially within the most valuable first few frames of an exposure, thus counteracting any potential benefit of better cryoprotection that liquid-helium cooling may offer for single-particle cryo-EM.

scholarly journals 314 FULL-TERM DEVELOPMENT OF RAT OOCYTES MICROINSEMINATED WITH FREEZE-DRIED SPERMATOZOA

2005 ◽  
Vol 17 (2) ◽  
pp. 307
Author(s):  
M. Hirabayashi ◽  
M. Kato ◽  
S. Hochi
Keyword(s):  
Liquid Nitrogen ◽  
Freeze Drying ◽  
Pure Water ◽  
Full Term ◽  
Female Rats ◽  
Membrane Disruption ◽  
Developmental Potential ◽  
Fresh Freeze ◽  
Freeze Dried

Since freeze-dried spermatozoa can be stored at ambient or refrigerated temperature, the costs required for maintenance and shipping of spermatozoa can be reduced. To date, viable offspring in mice (Wakayama and Yanagimachi 1998 Nat. Biotech. 16, 639) and rabbits (Liu et al. 2004 Biol. Reprod. 70, 1776) have been produced by intracytoplasmic sperm injection (ICSI) using freeze-dried samples. The objectives of the present study were to examine whether freeze-dried rat spermatozoa can participate in full-term development by ICSI, and whether sonication prior to freeze-drying of the spermatozoa influences the offspring rate. Spermatozoa from cauda epididymides of Sprague-Dawley (SD) rats were collected in 10 mM TRIS/HCl buffer supplemented with 50 mM NaCl and 50 mM EGTA. A 2 × 3 factorial-designed experiment was conducted. The sperm suspensions were either sonicated for 10 s using a 10% power output from an ultrasonic cell disruptor or not sonicated. The sperm suspensions were then processed for freeze-thawing (100-μL sample in 1.0-mL cryotube was cooled in liquid nitrogen vapor, stored at -196°C for 48 h, and thawed in a 25°C water bath) and freeze-drying (100-μL sample in 1.5-mL polypropylene tube was frozen in liquid nitrogen for 20 s, lyophilized for 6 h by a freeze-drying apparatus, stored at 4°C for 48 h, and rehydrated with 100 μL ultra pure water), or were subjected to immediate use for ICSI. The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4 μm in diameter, as described previously (Hirabayashi et al. 2002 Transgenic Res. 11, 221). The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. The in vivo developmental potential of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa is shown in the table below. Viable rat offspring were produced in all six experimental groups, with the offspring rates at 2.5–35.0%. Sonication treatment of rat spermatozoa to induce membrane disruption and tail/midpiece dissociation from the heads was effective in increasing the offspring rate after ICSI. The positive effect of sperm sonication may be explained as facilitating decondensation of sperm heads by membrane disruption in the spontaneously activating rat oocytes. Thus, successful participation of freeze-dried rat spermatozoa into full-term development was demonstrated by applying the ICSI. Table 1. In vivo development of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa

scholarly journals The Effect Of Hydrazine Addition On The Formation Of Oxygen Molecule By Fast Neutron Radiolysis

KnE Energy ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
G.R. Sunaryo
Keyword(s):  
Room Temperature ◽  
Pure Water ◽  
Water System ◽  
Calculation Model ◽  
High Dose ◽  
Primary Coolant ◽  
Batch System ◽  
Different Temperatures ◽  
Oxygenated Water ◽  
Deaerated Condition

<p>Hypothetically speaking, hydrazine could suppress the oxygen formation as a major of corrosion initiator. In this work, we developed a calculation model to understand the effect of hydrazine addition toward the oxygen under PWR condition. Our great interest is to study whether this strategy would also be effectively applied in PWRs<a href="file:///C:/Users/Mohamad%20Mostafa/Desktop/Knowledge%20E/In%20Press%20Conferences/ICoNETS-2015/Source-Manuscripts/20_L05-Geni_p136-141.docx#_msocom_1">[P1]</a> . In the present work, the effect of hydrazine on suppressing the molecule oxygen under neutron irradiation is described.  The simulation was done by using FACSIMILE.  The variation dose applied assuming a batch system and at high dose ~10<sup>4</sup> Gy s<sup>-1</sup>.  Three different temperatures were applied, which are room temperature, 250 and 300 <sup>o</sup>C at two system oxygenated water, which are aeration and deaeration. At room temperature, for deaerated condition, added hydrazine under a range of 10<sup>-6</sup> – 10<sup>-4</sup> M into primary coolant were not effective to suppress  O<sub>2 </sub>form since the effect was similar as in the pure water system since for 10<sup>-3</sup> M hydrazine addition, a large produce of O<sub>2 </sub>were obtained. In reverse, for deaerated condition, hydrazine concentrate about 10<sup>-3</sup> M can suppress O<sub>2</sub> form significantly, while hydrazine add in the range between 10<sup>-6</sup> – 10<sup>-4</sup> M is again confirmed to be the same as in pure water system. For high temperature, at 250 and 300 <sup>o</sup>C, the results showed that in deaerated condition, hydrazine addition can suppress  O<sub>2  </sub>form<sub> </sub>proportionally to its concentration while in aerated condition, hydrazine add with concentration of 10<sup>-6</sup> and 10<sup>-5</sup> M were not effectively to suppress O<sub>2  </sub>form,<sub> </sub>a slightly decrease of O<sub>2</sub> occurred due to the addition of 10<sup>-4</sup> M hydrazine and 10<sup>-3</sup> M of hydrazine can suppress the formation of O<sub>2</sub> significantly. <a href="file:///C:/Users/Mohamad%20Mostafa/Desktop/Knowledge%20E/In%20Press%20Conferences/ICoNETS-2015/Source-Manuscripts/20_L05-Geni_p136-141.docx#_msocom_2">[P2]</a> </p><div><hr align="left" size="1" width="33%" /><div><div><p> <a href="file:///C:/Users/Mohamad%20Mostafa/Desktop/Knowledge%20E/In%20Press%20Conferences/ICoNETS-2015/Source-Manuscripts/20_L05-Geni_p136-141.docx#_msoanchor_1">[P1]</a>The added sentence</p><p> </p></div></div><div><div><p> <a href="file:///C:/Users/Mohamad%20Mostafa/Desktop/Knowledge%20E/In%20Press%20Conferences/ICoNETS-2015/Source-Manuscripts/20_L05-Geni_p136-141.docx#_msoanchor_2">[P2]</a>The revised sentence</p></div></div></div>

scholarly journals Inverse Leidenfrost Effect: Levitating Drops on Liquid Nitrogen

Langmuir ◽  
2016 ◽  
Vol 32 (17) ◽  
pp. 4179-4188 ◽  
Author(s):  
M. Adda-Bedia ◽  
S. Kumar ◽  
F. Lechenault ◽  
S. Moulinet ◽  
M. Schillaci ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Leidenfrost Effect

Fraud risk factors and tendency to commit fraud: analysis of employees’ perceptions

2019 ◽  
Vol 35 (4) ◽  
pp. 545-557
Author(s):  
Jesi Rizky Anindya ◽  
Desi Adhariani
Keyword(s):  
Risk Factors ◽  
Sampling Method ◽  
Small Samples ◽  
Dominant Factor ◽  
Fraud Triangle ◽  
Fraud Prevention ◽  
Content Type ◽  
Unethical Conduct ◽  
Fraud Risk ◽  
Convenience Sampling

Purpose This study aims to determine the fraud risk factors perceived by employees to have the greatest influence on individuals committing fraud as an unethical conduct, as well as to analyze employees’ opinions on fraud prevention program. Design/methodology/approach The fraud risk factors in this study are based on the concept of the fraud triangle as developed by Donald Cressey, as well as examples of situations set out in SAS No. 99. The samples used in this study are company employees who have been selected using the convenience sampling method. Findings A survey of 109 employees reports that none of the three factors (pressure, opportunity and rationalization) has a significant influence on fraud. However, when comparing the factors, the pressure is considered to have the highest impact. In terms of fraud prevention, the employees suggest that it is extremely important to implement all prevention tools, especially with regard to the adequate segregation of duties. Research limitations/implications Limitations of this study in terms of method and small samples are expected to inform future studies to overcome the limitations by using other methods such as interview and by collecting more respondents to gather their perceptions and opinions. Originality/value This study contributed to the literature in confirming the pressure as the dominant factor and in confirming the importance of anti-fraud programs as suggested by the agency theory.

How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

2019 ◽  
Vol 48 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Energy Use ◽  
Optimal Choice ◽  
Structural Basis ◽  
Detective Quantum Efficiency ◽  
Quantum Metrology ◽  
Cryo Electron Microscopy ◽  
Induced Motion

Impressive though the achievements of single-particle cryo–electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards ( a) the detective quantum efficiency of cameras at high resolution, ( b) converting phase modulations to intensity modulations in the image, and ( c) recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology. With the help of such improvements, it does not seem to be too much to imagine that determining the structural basis for every aspect of catalytic control, signaling, and regulation, in any type of cell of interest, could easily be accelerated fivefold or more.

Inverse Leidenfrost Effect of Cryoprotectant Droplets on Liquid Nitrogen Surface

Cryobiology ◽  
2018 ◽  
Vol 80 ◽  
pp. 184
Author(s):  
Haikao Feng ◽  
Yi Xu ◽  
Tingting Yang
Keyword(s):  
Liquid Nitrogen ◽  
Leidenfrost Effect
Sign in / Sign up

Export Citation Format

Share Document