Controlled Environment Vitrification System Research Articles

Development of an Organic Controlled Environment Vitrification System to Expand Cryo-Protection of Beam Sensitive Materials

Development of an Organic Controlled Environment Vitrification System to Expand Cryo-Protection of Beam Sensitive Materials

Nanostructure Formation in the Lecithin/Isooctane/Water System

We present here for the first time a study of the self-assembled nanostructures in the lecithin/isooctane/water system by direct-imaging techniques, namely, cryogenic transmission electron microscopy (cryo-TEM) and cryogenic scanning electron microscopy (cryo-SEM). Along the dilution line [water]/[lecithin] = 5, we identified a nanostructural development with the increase of lecithin concentration. The system changes from a single reverse micellar phase, through a reverse micellar phase coexisting with a lamellar phase, and finally to a reverse liquid crystalline cubic phase and a lamellar phase. We compared the nanostructures formed when phosphatidylcholine rather than naturally occurring lecithin is used and found that both phase behavior and nanostructure are significantly different. The use of the two complementary cryo-EM techniques proved very efficient in the nanostructural characterization of the system. We also performed small-angle X-ray scattering to confirm our findings. Since the system is very sensitive to changes in composition, the cryo-EM specimens were prepared in a Controlled Environment Vitrification System (CEVS) that has been modified for our specimen preparation needs. We were able to overcome the challenges involved in directly imaging this nonaqueous (oil-rich), concentrated complex liquid systems, thus extending the usefulness of those characterization techniques.

Imaging New Transient Nanostructures Using a Microfluidic Chip Integrated with a Controlled Environment Vitrification System for Cryogenic Transmission Electron Microscopy

Nanostructures (vesicles, micelles, bilayers) are important in nanomedicine and biochemical processes. They are agents for encapsulation and eventual release of drugs, flavors, and fragrances. The structural transition from micelles to vesicles through disk-like intermediate states has been demonstrated previously. Here, we disclose a new route for the micelle-vesicle transition, where micelles aggregate to first form long tubules that become unstable, and break up into vesicles. A simple theory, based on energy principles, is presented to explain the tubule-vesicle transition. Observation of this new tubular intermediate state has been facilitated by the development of an integrated microfluidic chip/cryogenic transmission electron microscopy (cryo-TEM) unit. Although this transition has been observed in a specific amphiphilic system where micellar solutions of cetyltrimethylammonium bromide (CTAB) and dodecylbenzene sulfonic acid (HDBS) are mixed to form vesicles, this new tool can be applied broadly to study transient structures in nanoscale systems under the very controlled conditions provided by microfluidics.

New controlled environment vitrification system for preparing wet samples for cryo‐SEM

A new controlled environment vitrification system (CEVS) has been designed and constructed to facilitate examination by cryogenic scanning electron microscopy (Cryo-SEM) of initial suspension state and of microstructure development in latex, latex-composite and other coatings while they still contain solvent. The new system has a main chamber with provisions for coating as well as drying, and for well-controlled plunging into cryogen. An added subsidiary chamber holds samples for drying or annealing over minutes to days before they are returned to the main chamber and plunged from it. In the main chamber, samples are blade-coated on 5 x 7 mm pieces of silicon wafer and held at selected temperature and humidity for successively longer times, either there or after transfer along a rail into the subsidiary chamber. They are then placed in the sample holder mounted on the plunge rod, so as to permit adjustment of the sample's attitude when it plunges, at controlled speed, into liquid ethane at its freezing point, to a chosen depth, in order to solidify the sample without significant shear or freezing artifacts. The entries of plunging samples and related sample holders into liquid ethane were recorded with a high-speed, high-resolution Photron digital camera. The data were interpreted with a new hypothesis about the width of the band of extremely rapid cooling by deeply subcooled nucleate boiling below the line of entry. Complementary cryo-SEM images revealed that the freezing rate and surface shearing of a sample need to be balanced by adjusting the plunging attitude.

Towards understanding selenocysteine incorporation into bacterial proteins

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.

New controlled environment vitrification system for cryo-transmission electron microscopy: design and application to surfactant solutions.

A newly designed controlled environment vitrification system for cryo-transmission electron microscopy of vitrified samples (cryo TEM) is presented. It significantly reduces the lag-time from blotting to vitrification by means of an optimized blotting procedure. Furthermore, a modified transfer system allows rapid transfer of the vitrified sample into the microscope and reduces the equilibration time. The practicable throughput of samples is thereby markedly increased. This new equipment is used to determine the overall size of individual polymer-like mixed lecithin-bile salt micelles, which very critically depends on the composition of the sample. The quantitative agreement with the results from independent light scattering experiments on the same sample supports the validity and performance of the presented sample preparation and handling procedures. In addition, these experiments demonstrate that cryo TEM experiments not only provide direct structural information, but furthermore permit a quantitative determination of micellar properties.

EFTEM Elemental Mapping of Particles Frozen in Amorphous Solutions

The preparation of frozen amorphous nano-meter thin films of solutions containing particles, and imaging with energy filtered TEM, opens for the first time the possibility to map the elemental distribution in solids interacting with liquids. The future possible areas of research in solution include: dynamic of crystal growth processes, adsorption mechanisms, ion exchange, structure determination of nano-sized solids in equilibrium with different solvents, etc.The sample preparation uses a controlled-environment vitrification system. Solidification of the liquid phase was achieved by plunging the solution, suspended in the holes of a lacy carbon on a copper grid, into liquid ethane (-174 °C). The specimen was transferred into the microscope by an Oxford CT3500 cryo-holder. The specimen temperature in the microscope column can be kept at -183 °C. The thickness of the vitrified film's, including the particles, should be about 50-100 nm or preferably less. The Philips CM120 BioTWIN Cryo microscope, here used for cryo-EFTEM, has a focal length of 6 nun and a structural resolution of 0.4 nm.

Solubilization of Nonionic Monoalkyl Amphiphile−Cholesterol Vesicles by Octyl Glucoside: Cryo-Transmission Electron Microscopy of the Intermediate Structures

The micellization by octyl glucoside (OG) of diglycerol hexadecyl ether−cholesterol (1:1 wt %) sonicated vesicles containing a small amount of dicetyl phosphate has been studied by quasielastic light scattering, turbidity measurement, and cryogenic transmission electron microscopy. By using a controlled environment vitrification system (CEVS), this last technique provides new information about the vesicle−micelle transition structures without introducing artifacts due to changes in temperature or saturation. The intermediate structures have been visualized as a function of increasing surfactant composition. In the 0−0.5 range of the OG to lipid molecular ratio in the aggregates, profound vesicle rearrangements, mainly fusion and deformation processes, occur. At higher OG concentrations, the vesicles significantly decrease in size and form clusters which originate from a dense precipitate and coexist with mixed micelles, the proportion of lamellar structures being progressively reduced in favor of micelliz...

Stabilizing aqueous clay suspensions by AOT vesicular solutions: a cryo-TEM study

Production, storage, and performance of suspensions of colloidal solid particles depend critically on suspensions stability. Past studies mostly focused on roles of attractive van der waals force and repulsive electrostatic force between particles. In contrast, this paper reports that aqueous suspensions of clay particles of maximum dimension of 5 to 20 nm can be greatly stabilized by adding vesicular solutions of the anionic surfactant Aerosol OT (AOT).How does the interaction between the clay particles, which bear charge, and multiple bilayers of AOT vesicles lead to more stable suspensions? Cryogenic transmission electron microscopy (cryo-TEM) was brought to bear on this question because it can capture the microstructure in fluids without staining or drying artifacts. Cryo-TEM samples were made by rapidly freezing thin liquid films (200-500 nm) in a controlled environment vitrification system (CEVS)5 to preserve the original structures. Clay particles (often hexagonal) were flat plates and tended to associate face-to-face (Fig. la). Cryo-TEM images show that 3 wt% and 5 wt% AOT formed unilamellar and multilamellar vesicles in solutions (Fig. lb-c).

Cryo-TEM of amphiphilic polymer and amphiphile/polymer solutions

To achieve complete microstructural characterization of self-aggregating systems, one needs direct images in addition to quantitative information from non-imaging, e.g., scattering or Theological measurements, techniques. Cryo-TEM enables us to image fluid microstructures at better than one nanometer resolution, with minimal specimen preparation artifacts. Direct images are used to determine the “building blocks” of the fluid microstructure; these are used to build reliable physical models with which quantitative information from techniques such as small-angle x-ray or neutron scattering can be analyzed.To prepare vitrified specimens of microstructured fluids, we have developed the Controlled Environment Vitrification System (CEVS), that enables us to prepare samples under controlled temperature and humidity conditions, thus minimizing microstructural rearrangement due to volatile evaporation or temperature changes. The CEVS may be used to trigger on-the-grid processes to induce formation of new phases, or to study intermediate, transient structures during change of phase (“time-resolved cryo-TEM”). Recently we have developed a new CEVS, where temperature and humidity are controlled by continuous flow of a mixture of humidified and dry air streams.

Cryo-TEM of micelles and lyotropic liquid crystals in non-aqueous solvents

Surfactant molecules can form spherical micelles in water above the critical micellization concentration. At still higher concentrations, they can form thread-like micelles and liquid crystals. Cryo-transmission electron microscopy of these microstructures in water is now used successfully for studying aqueous biological and synthetic systems. The controlled environment vitrification system (CEVS) makes it possible to prepare samples accurately. Further, The on-grid processing makes specimens possible from evolving systems halted in mid-process.Despite all the successes with aqueous systems, imaging of microstructures formed in non-aqueous solvents has not been reported. The main difficulties are radiation damage, thermal drift of the specimen, and low contrast. As small a spot size and as well spread a beam as possible are needed for low magnification searching. During the image recording, the minimum dose and lowest magnification possible are desired in order to avoid the thermal drift and radiation damage while maintaining an adequate signal-to-noise ratio.

The structure of membranes and membrane proteins embedded in amorphous ice

TEM imaging of frozen-hydrated lipid vesicles has been done by several groups Thermotrophic and lyotrophic polymorphism has been reported. By using image processing, computer simulation and tilt experiments, we tried to learn about the influence of freezing-stress and defocus artifacts on the lipid polymorphism and fine structure of the bilayer profile. We show integrated membrane proteins do modulate the bilayer structure and the morphology of the vesicles.Phase transitions of DMPC vesicles were visualized after freezing under equilibrium conditions at different temperatures in a controlled-environment vitrification system. Below the main phase transition temperature of 24°C (Fig. 1), vesicles show a facetted appearance due to the quasicrystalline areas. A gradual increase in temperature leads to melting processes with different morphology in the bilayer profile. Far above the phase transition temperature the bilayer profile is still present. In the band-pass-filtered images (Fig. 2) no significant change in the width of the bilayer profile is visible.

A cryogenic transmission electron microscopy study of counterion effects on hexadecyltrimethylammonium dichlorobenzoate micelles

We have used the controlled environment vitrification system to prepare vitrified TEM specimens of aqueous solutions of hexadecyltrimethylammonium 3,5-dichlorobenzoate (CTA−3,5−ClBz) and hexadecyltrimethylammonium 2,6-dichlorobenzoate (CTA−2,6ClBz) at various concentrations. We report direct images of the cylindrical micelles of CTA−3,5ClBz at all concentrations studied

On-the-Grid Processing and Time-Resolved Cryo-TEM: The Extension of the Thin Liquid Film Vitrification Method

The thin vitrified liquid film technique was pioneered by Dubochet and co-workers. The technique is based on forming a thin liquid film of the system to be examined over a holey carbon film, and plunging it, using a mechanical plunger, into liquid ethane at its freezing point. Talmon and co-workers have developed the controlled environment vitrification system (CEVS), that makes it possible to prepare the sample under controlled conditions of temperature and saturation with volatiles. This has extended the applicability of the technique to labile systems that are sensitive to temperature and concentration changes, and to the study of microstructures that exist above (up to 95 °C), and below (down to −20 °C) room temperature. Here we discuss two extensions to cryo-TEM specimen preparation by thin fluid film vitrification, both based on specimen preparation in the CEVS: on-the-grid processing and time-resolved cryo-transmission electron microscopy.

A temperature-jump technique for time-resolved cryo-transmission Electron Microscopy

Transmission electron microscopy of rapidly-frozen, hydrated specimens (cryo-TEM) is a powerful way of examining labile microstructures. This technique avoids some artifacts associated with conventional preparative methods. Use of a controlled environment vitrification system (CEVS) for specimen preparation reduces the risk of unwanted sample changes due to evaporation, and permits the examination of specimens vitrified from a defined temperature. Studies of dynamic processes with time resolution on the order of seconds, in which the process was initiated by changes in sample pH, have been conducted. We now report the development of an optical method for increasing specimen temperature immediately before vitrification. Using our method, processes that are regulated by temperature can be initiated in less than 500 msec on the specimen grid. The ensuing events can then be captured by plunge-freezing within an additional 200 msec.Dimyristoylphosphatidylcholine (DMPC) liposomes, produced by extrusion, were used as test specimens. DMPC undergoes a gel/liquid crystalline transition at 24°C, inducing a change in liposome morphology from polyhedral to spherical. Five-μl aliquots of DMPC dispersions were placed on holey-carbon-filmed copper grids mounted in the CEVS environmental chamber, and maintained at 6-8°C and 80% relative humidity. Immediately before the temperature jump most of the sample was blotted away with filter paper, leaving a thin specimen film on the grid. Upon pressing the trigger, an electronic control circuit generated this timed sequence of events. First, a solenoid-activated shutter was opened to heat the specimen by exposing it for a variable time to the focused beam of a 75W Xenon arc lamp. Simultaneously, a solenoid-activated cryogen shutter in the bottom of the CEVS was opened. Next, the lamp shutter was closed after the desired heating interval. Finally, a solenoid-activated cable release was used to trigger a spring-loaded plunger in the CEVS, propelling the sample into a reservoir of liquid ethane. Vitrified samples were subsequently transferred to a Zeiss EM902 TEM, operated in zero-loss brightfield mode, for examination at −163°C.

Controlled environment vitrification system: An improved sample preparation technique

The controlled environment vitrification system (CEVS) permits cryofixation of hydrated biological and colloidal dispersions and aggregates from a temperature- and saturation-controlled environment. Otherwise, specimens prepared in an uncontrolled laboratory atmosphere are subject to evaporation and heat transfer, which may introduce artifacts caused by concentration, pH, ionic strength, and temperature changes. Moreover, it is difficult to fix and examine the microstructure of systems at temperatures other than ambient (e.g., biological systems at in vivo conditions and colloidal systems above room temperature). A system has been developed that ensures that a liquid or partially liquid specimen is maintained in its original state while it is being prepared before vitrification and, once prepared, is vitrified with little alteration of its microstructure. A controlled environment is provided within a chamber where temperature and chemical activity of volatile components can be controlled while the specimen is being prepared. The specimen grid is mounted on a plunger, and a synchronous shutter is opened almost simultaneously with the release of the plunger, so that the specimen is propelled abruptly through the shutter opening into a cryogenic bath. We describe the system and its use and illustrate the value of the technique with TEM micrographs of surfactant microstructures in which specimen preparation artifacts were avoided. We also discuss applications to other instruments like SEM, to other techniques like freeze-fracture, and to novel "on the grid" experiments that make it possible to freeze successive instants of dynamic processes such as membrane fusion, chemical reactions, and phase transitions.

Freeze-fracture-replication using the controlled environment vitrification system (CEVS).

Freeze-fracture-replication using the controlled environment vitrification system (CEVS).

Freezing Dynamical Sol-gel processes with the controlled environment vitrification system (CEVS)

A sol is a stable suspension of submicron particles in a liquid while a gel is a three dimensional network of these particles which is bicontinuous with the liquid phase. Sol particles are stabilized by surface charges or by steric hindrance of molecules on the particle surface. Sols can be gelled by the agglomeration of the particles; for example, by reducing the inter-particle repulsion with the addition of a salt. The sol-gel transformation is an important industrial, biological and household process. Novel ceramic processing techniques, blood-clotting, and white-sauce manufacturing are some diverse examples. Therefore, there has been extensive interest in relating the gel structure to changes in the physical properties that occur during gelation.Direct observation of thin vitrified films is the preferred technique for microstructural determination of colloidal dispersions in the size range of 1-1000nm.

Preparation of Liquids and Colloidal Dispersions for Tem: Cryo-Sem Studies of Bare-Film and Freeze-Fracture Specimens

ABSTRACTLiquid and colloidal microstructures can be imaged at high resolution with cryo-TEM and cryo-SEM by fast-freezing with a Controlled Environment Vitrification System. Frozen bare-films can be directly imaged by TEM, while films sandwiched between metal plates can be frozen, fractured, and directly imaged with cryo-SEM or indirectly imaged by TEM after replication. We describe sample preparation techniques and report cryo-SEM studies of particle distributions and fracture patterns in such specimens.