A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography

The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has long been hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-defined images to be recorded. A new "G2 Polara" microscope from FEI Company was used to image various biological samples cooled by either liquid nitrogen or liquid helium to approximately 82 or approximately 12 K, respectively, and the results were compared with particular interest in the doses (10-200 e-/A2) and resolutions (3-8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at approximately 12K through the first several tens of e-/A2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no difference in the preservation of medium-resolution (3-5 nm) structural detail at the two temperatures. Tomographic reconstructions produced with total doses between 10 and 350 e-/A2 showed better results at approximately 82 K than approximately 12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens.

We have carried out a theoretical analysis of specimen cooling rate under ideal conditions during impact freezing and liquid-jet freezing. The analysis shows that use of liquid helium instead of liquid nitrogen as cooling medium during impact freezing results in an increase in a specimen cooling rate of no more than 30-40%. We have further shown that when both impact freezing and liquid-jet freezing are conducted at liquid nitrogen temperature, the two methods give approximately the same specimen cooling rate under ideal conditions except for a thin outer layer of the specimen. In this region impact freezing yields the highest cooling rate.     

Methods and Principles of Fixation by Freeze-Substitution

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O°C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175°C. (with precautions). The frozen tissue was placed in substituting fluid at -70°C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.     

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.

Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at −78°. They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.

Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below −60°. Those cells at intermediate temperatures from −20° to −45°, were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at −30°. The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below −60° or into liquid nitrogen.

The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from −70° to −10° before rapid rewarming. There was little damage when samples were held at temperatures below −50° for 10 minutes or below −60° for 16 hours. No cavities were found in these cells. Above −45°, and especially at −30°, however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

     

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from −126 °C to −121 °C, and devitrification was initiated at −109 °C. Interestingly, samples entered rubbery state at −121 °C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (−196 °C), and two temperatures of liquid nitrogen vapors within the dewar (−50 °C and −70 °C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30 seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.     

The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels

During long-term cryopreservation, ice sediment accumulates in storage Dewars and poses a risk of microbial contamination to stored samples. Ice accumulates in liquid nitrogen via two general processes: (1) ice forming in the atmosphere above an open Dewar falls into the vessel; and (2) ice forming on cold surfaces of the Dewar or inventory system enters the liquid nitrogen. These ice crystals aggregate and entrap other materials, such as bacteria, fungal spores, and general laboratory debris present within the liquid nitrogen. Measured changes in the ultrastructure of ice aggregates following long-term storage are consistent with transient warming events to temperatures of -100 degrees C. Bacteria were identified in all samples and filamentous fungi in 9 out of 10 samples. These micro-organisms are commonly found in the environment and would not be expected to have been derived from IVF samples. Some of the bacteria identified are associated with nosocomial infections in humans. The implications that the association of microbial contamination with ice crystals has on cryopreservation procedures are discussed.     

Ultrastructural and thermocouple evaluation of rapid freezing techniques

The quality of freeze-fixation for electron microscopy is dependent upon the size of intracellular ice crystals. In the absence of cryoprotectants, ice crystal growth is thought to be related to the speed with which the specimen is cooled. The purpose of this study was to investigate the relationship between the cooling rate and ultrastructural preservation in commonly used freezing techniques. The techniques studied included immersion in stirred and unstirred forms of five quenching fluids: liquid nitrogen, isopentane, Freon 12, Freon 22, and propane. Also studied were freezing in a flowing stream of coolant using liquid nitrogen and liquid helium and freezing on a metal surface using cooper and mercury chilled to liquid nitrogen temperature. For each technique a cooling curve was obtained with a 0.360-mm thermocouple which was dropped into the quenching fluids or brought into contact with the metal surfaces. From oscilloscope tracings, the cooling rates were determined in degrees centigrade per second to −100 °C. To evaluate ultrastructural preservation 0.5-mm-thick slices of rat kidney were frozen by each of the techniques and dried in an all glass freeze-drier. The final evaluation was made from electron micrographs of the best morphological preservation yielded by each technique. The results indicate that the copper and mercury surfaces and propane gave the highest cooling rates and the best morphological preservation. The other techniques cooled at decreasing rates and correspondingly showed decreasing abilities to preserve ultrastructure. This work demonstrates that the preservation of cellular ultrastructure by freezing is dependent upon the cooling rate and that as the cooling rate is increased, ultrastructural preservation is enhanced.     

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.     

A helium gas probe for use in cryosurgery

The design and testing of a prototype cryosurgical probe utilizing helium gas precooled with liquid nitrogen are described. An 8-mm-diameter probe produced an ice ball with a diameter of 28 mm after 10 min freezing using a helium gas flow rate of 42 liter/min. This indicated a surface heat transfer coefficient of 0.34 W/cm² °K and temperature of ?138 °C at the probe tip. Improved performance figures can be achieved using higher gas pressures and flow rates. A helium gas flow system schematic for use with this new type of cryoprobe is also presented. It is claimed that this system will overcome the problems of developing both multiple-tipped probes and small-diameter needle probes for use in cryoanalgesia.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

Ice nucleation and freezing in undercooled cells

DSC has been employed to study the effect of cooling on a range of cells under exclusion of extracellular ice and in the absence of chemical cryoprotectants. In contrast to earlier reports, all the cells studied were found to freeze at temperatures above that indicated for homogeneous nucleation of ice in undercooled liquid water. In the case of human erythrocytes this temperature difference was only 0.5 °, but for yeast cells and cells of plant origin the difference amounted to ?9 °.Nucleation of ice within the cell (or at the cell wall/membrane) must therefore be initiated by a heterogeneous mechanism. A kinetic analysis of the temperature dependence of nucleation shows the rates to be consistent with the dimensions of the plant cells (or organelles), if these were to be the active nucleators. However, the nucleation kinetics of human erythrocytes are extremely temperature sensitive, and the kinetic parameters only differ by small, though significant, extents from those of the suspension medium.Possible nucleation mechanisms are discussed in terms of the experimental data and the cell dimensions. Finally, one of the underlying assumptions of the kinetic analysis, i.e., that ice growth must be rapid compared to nucleation, has been tested and validated by freeze-fracture electron microscopy.     

Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K

In an attempt to determine why cooling with liquid helium actually proved disadvantageous in our electron cryotomography experiments, further tests were performed to explore the differences in vitreous ice at approximately 82 and approximately 12 K. Electron diffraction patterns showed clearly that the vitreous ice of interest in biological electron cryomicroscopy (i.e., plunge-frozen, buffered protein solutions) does indeed collapse into a higher density phase when irradiated with as few as 2-3 e-/A2 at approximately 12 K. The high density phase spontaneously expanded back to a state resembling the original, low density phase over a period of hours at approximately 82 K. Movements of gold fiducials and changes in the lengths of tunnels drilled through the ice confirmed these phase changes, and also revealed gross changes in the concavity of the ice layer spanning circular holes in the carbon support. Brief warmup-cooldown cycles from approximately 12 to approximately 82 K and back, as would be required by the flip-flop cryorotation stage, did not induce a global phase change, but did allow certain local strains to relax. Several observations including the rates of tunnel collapse and the production of beam footprints suggested that the high density phase flows more readily in response to irradiation. Finally, the patterns of bubbling were different at the two temperatures. It is concluded that the collapse of vitreous ice at approximately 12 K around macromolecules is too rapid to account alone for the problematic loss of contrast seen, which must instead be due to secondary effects such as changes in the mobility of radiolytic fragments and water.     

Transporting Cells in Semi-Solid Gel Condition and at Ambient Temperature

Mammalian cells including human cancer cells are usually transported in cryovials on dry ice or in a liquid nitrogen vapor shipping vessel between different places at long distance. The hazardous nature of dry ice and liquid nitrogen, and the associated high shipping cost strongly limit their routine use. In this study, we tested the viability and properties of cells after being preserved or shipped over long distance in Matrigel mixture for different days. Our results showed that cells mixed with Matrigel at suitable ratios maintained excellent viability (>90%) for one week at room temperature and preserved the properties such as morphology, drug sensitivity and metabolism well, which was comparable to cells cryopreserved in liquid nitrogen. We also sent cells in the Matrigel mixture via FedEx service to different places at ambient temperature. Upon arrival, it was found that over 90% of the cells were viable and grew well after replating. These data collectively suggested that our Matrigel-based method was highly convenient for shipping live cells for long distances in semi-solid gel condition and at ambient temperature.     

Freezing and cryoprotective dehydration in an Antarctic nematode (Panagrolaimus davidi) visualised using a freeze substitution technique

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).     

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used "negative stains". Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low dose imaging techniques to avoiding radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Microbial metabolism in ice and brine at -5°C

Metabolic activity, but not growth, has been observed in ice at temperatures from -5°C to -32°C. To improve understanding of metabolism in ice, we simultaneously examined various aspects of metabolism ((14) C-acetate utilization, macromolecule syntheses and viability via reduction of CTC) of the glacial isolates Sporosarcina sp. B5 and Chryseobacterium sp. V3519-10 during incubation in nutrient-rich ice and brine at -5°C for 50 days. Measured rates of acetate utilization and macromolecule syntheses were high in the first 20 days suggesting adjustment to the lower temperatures and higher salt concentrations of both the liquid vein network in the ice and the brine. Following this adjustment, reproductive growth of both organisms was evident in brine, and suggested for Sporosarcina sp. B5 in ice by increases in cell numbers and biomass. Chryseobacterium sp. V3519-10 cells incubated in ice remained active. These data indicate that neither low temperature nor high salt concentrations prohibit growth in ice, but some other aspect of living within ice slows growth to within the detection limits of current methodologies. These results imply that microbial growth is plausible in natural ice systems with comparable temperatures and sufficient nutrients, such as debris-rich basal ices of glaciers and ice masses.     

Survival of Plant Tissue at Super-Low Temperatures. IV. Cell Survival with Rapid Cooling and Rewarming

Thin unmounted cortical tissue sections from winter twigs of the mulberry tree were held with a thin forceps and rapidly immersed in liquid nitrogen from room temperatures without prefreezing. They were rewarmed; rapidly in water at 10° to 40°, or slowly, in air at room temperatures. In those sections rapidly rewarmed, all survived. None survived in those sections rewarmed slowly in air.

Tissue sections mounted between coverglasses with water were extracellulary prefrozen at the temperatures low enough to dehydrate almost all of the freezable water in cells. These sufficiently prefrozen cells could survive immersion in liquid nitrogen, and the survival value was very little affected by the rates of cooling to and rewarming from super-low temperatures. With insufficient prefreezing at higher temperatures, however, the rewarming process seriously influenced the survival value of cells frozen at super-low temperatures. Slow rewarming in air destroyed all of the cells, while rapid rewarming in water at 30° did not affect them. An abrupt decrease in the survival value in insufficiently prefrozen cells during rewarming was also observed at temperatures above approximately −50° following immersion in liquid nitrogen. Very little decrease in the survival value was observed in any of the cells that had been sufficiently prefrozen.

These results indicate that cells which are insufficiently prefrozen may contain freezable water which nucleates during rapid cooling in liquid nitrogen and then grows during the subsequent slow rewarming into ice masses which destroy the viability of the cells. Such fatal intracellular freezing rarely occurs in sufficiently prefrozen cells, irrespective of the rate of cooling to or rewarming from super-low temperatures.

     

Freeze-fixation at high subzero temperatures.

Previous attempts to determine the distribution of ice in frozen tissues at high sub-zero temperatures generally called for the further cooling of the tissues in question to facilitate freeze-drying, freeze-substitution, and freeze-fracture replication. Direct cryomicroscopic determinations, free from uncertainties stemming from changes in sample temperature could, it seemed, only be made in certain special cases. We have presented an isothermal “freeze-fixation” procedure designed to permit, instead, the postthaw retention of the freezing pattern and the conventional processing, afterward, of the thawed specimen. The method demands the exposure of the frozen tissues to fixative solutions incapable of dissolving ice. Frozen specimens are immersed in aqueous fixative solutions prepared in each instance (1) to freeze at a temperature equal to that at which fixation is to be conducted, (2) to contain quantities of finely divided ice sufficient to guarantee the maintenance of a constant water activity. Frozen frog and rat hearts and skeletal muscle tissues were exposed to formaldehyde, formaldehyde/ glutaraldehyde, and glutaraldehyde solutions at ?2, ?5, and ?10 °C, the temperatures being maintained in each case to ± 0.1 °C, or better. Tissues withdrawn at intervals were thawed, postfixed, dehydrated, embedded, and sectioned. The sections demonstrated the retention, after thawing, of structural features characteristic of the frozen state. The small hearts we exposed to formaldehyde were fixed throughout in 3 hr at ?2 ° and in 20 hr at ?5 °C. The action of osmium tetroxide was investigated. The method appears to be well-suited to numerous experimental applications.