Cryoplunge™3 and Solarus® 950: a perfect duet for consistent, high quality frozen hydrated specimen preparations for cryo transmission electron microscopy (cryoEM)

CryoEM is a powerful tool in the arsenal of structural biologists and soft polymer chemists.  It is used to elucidate the high resolution structure of radiation-sensitive specimens such as dispersed biological macromolecular assemblies, 2D crystals and colloids. Key to the entire process of cryoEM is the preparation of the specimen. Cryoplunge3 (also known as Cp3) is Gatan’s new generation, semi-automatic plunge freezing instrument for the preparation of frozen hydrated specimens for cryoEM and, when paired with the Solarus 950 Advanced Plasma Cleaning System for preparing specimen support substrates, it is easy to produce consistent, high quality frozen hydrated specimens.

There are three main steps for preparing samples for cryoEM:

1. A small aliquot (approximately 3 µl) of a fluid suspension containing the sample is applied to the surface of a supporting substrate such as a holey (Quantifoil^® or C-flat™) or continuous carbon film that is attached to the surface of a standard TEM specimen grid.
2. The droplet is blotted with filter paper until only a thin layer (approximately 100 nm thick) is left on the support substrate.
3. The thin fluid layer is rapidly immersed into a suitable cryogen of high heat capacity, which gives instantaneous and contaminant-free freezing.

SEM image of Protochips C-flat ultra-flat holey carbon support CF-2/2-4C. Support hole pitch and diameter 2μm mounted on a 400 mesh copper EM grid. A uniform grid of 2μm holes facilitates automated data collection. Unlike most homemade and commercially available specimen supports, no plastic resin is used in the manufacturing process. Image compliments of Dr. David Nackashi, Protochips, Inc.

Preparing the specimen support 

The integrity of the specimen support film is important to the dispersal of the sample on the film, to the uniformity of ice thickness, and to the cleanliness of the resultant ice layer. Due to the fragility of support film, it is important that forces applied during the cryo specimen preparation procedure will not cause damage.  Preparing the specimen support usually involves making the surface of the support uniformly hydrophilic, hydrophobic or having a specific overall charge. While the surface of a support film, be it holey or continuous carbon, can be made hydrophobic by heating at 80 °C for about one hour, getting a uniformly hydrophilic surface requires some effort and potentially more expense, but with the outcome of higher a yield for data collection. 

Of the available methods that one can use to render the surface of the support film hydrophilic, plasma cleaning seems to provide the best results. The Solarus Model 950 is a state-of-the-art, versatile and easy to use advanced plasma cleaning system. It provides optimal plasma power for any cleaning condition by incorporating a real-time radio frequency (RF) auto-matching network. The RF power supply operates at settings of 10 to 65 Watts. The combination of the auto-matching RF and the highly accurate mass flow controllers (MFC) for hydrogen, oxygen and argon gas ensure stable plasma production during processing. The unique hydrogen and oxygen gas mixture recipe (patent pending) provides superior cleaning with less sputter damage for even the most fragile support films, and the specimen temperature rise in this plasma is lower than from other commonly used gas mixtures such as argon or argon/oxygen. As an example, Quantifoil or C-flat holey carbon films that they are positioned at the mid-point of the cleaning chamber can be rendered uniformly hydrophilic by using a 15 second process time with the hydrogen/oxygen gas mixture and an RF setting of 50 Watts. The free radicals formed when using the hydrogen/oxygen plasma break the bonds on the surface layer of the carbon to render the surface hydrophilic while minimizing sputter effects that could damage the carbon support. The spacious top entry specimen chamber is large enough to accommodate a pre-cleaned glass slide to support 50 or more grids at one time. Using the interactive touch-screen interface, each user can obtain consistent results by selecting one of seven factory programmed recipes for which the processing parameters have been optimized based on the particular type of specimen being cleaned. Plasma cleaning recipes can also be defined by the user.
 

The cleaning power of Solarus: The surface of a homemade holey carbon support film above was purposefully contaminated with oil by immersing it into a solution of 50 ml of acetone to which one drop of oil was added. It was allowed to dry at room temperature prior to insertion into the electron microscope. Two adjacent areas (a and b) were exposed to the electron beam for 20 and 60 seconds respectively to prove the presence of oil contamination on the surface of the support film. The formation of the fixed carbonaceous areas (dark disks) is the result of the condensed electron beam interacting with the oil on the surface of the support. The specimen holder, with support film intact, was then removed from the electron microscope and cleaned in the Solarus 950 Advanced Plasma Cleaning System for 30 seconds using the exclusive hydrogen/oxygen gas mixture to remove any remaining oil contamination (note that the previously exposed carbonaceous areas (a and b) remain fixed to the surface of the film.)  Following reinsertion into the microscope, a previously unexposed area (c) was exposed to the electron beam for 10 minutes.  Absence of the formation of a carbonaceous area indicates that any remaining oil contamination that was originally applied to the surface of the support was successfully removed by the plasma cleaning process. Experimental results and image courtesy of Dr. Steve Coyle, Gatan, Inc.

Blotting the specimen

The key to a good specimen is to obtain a uniformly thin layer of vitreous ice with the specimen embedded in it.  This is normally accomplished by blotting the specimen grid and fluid suspension using filter paper. Blotting the excess fluid can be done manually or by using an automated blotting device. Often with manual blotting, the thickness of the vitreous ice layer can be quite variable and a successful outcome depends very much on the skill of the individual doing the blotting. Commercially-available, automated and semi-automated plungers are more expensive but provide features such as timed, reproducible blotting and plunging within a high humidity chamber. Such features reduce variability and ensure consistent results from one user to the next. The automated plungers can translate into higher throughput in collecting high resolution cryoEM data.

The high humidity chamber of Cp3 maintains the freshly formed thin film until it is vitrified. A simply designed humidity wand with a replaceable cellulose sponge increases the humidity to approximately 98% fifteen minutes after insertion into the chamber. A humidity/temperature sensor connects to a built in meter allowing the atmospheric conditions within the chamber to be monitored at all times. This chamber can be removed to facilitate cleaning of its inner surface.  There are four blotting positions on the blot assemblies of Cp3 to match the four grid storage positions of the cryo grid box.  Frequent exchange of the filter paper helps to maintain consistency in blotting by avoiding oversaturation of the blotting paper in the high humidity of the chamber.

Blotting action of Cp3:  The adjustable pneumatic blotting action of Cp3 is triggered by an electronic timer that is accurate to 0.1 seconds. Cp3 comes with two interchangeable blot assemblies for two sided blotting (a) of the specimen grid and one blanking plug which provides the capability for one-sided front or back side blotting (b).  By setting the blot timer to zero, one can override the automatic blotting action altogether to facilitate other methods for applying the specimen which may not require blotting. The main specimen loading port is in the front of the chamber (c) but the specimen can also be applied to the specimen grid from either the left or right hand side by removing one of the blot assemblies (d).  A specially designed die cutter (e) allows the user to cut the filter paper of their choice for blotting the specimen. An easy to clean filter paper loading jig (f-h) ensures a virtually contamination free means for loading the blotting filter paper disks onto the blot assemblies. 

Plunging the specimen

Once the thin film containing the specimen is produced, it is immediately plunged into a suitable cryogen.  An example of a cryogen that is commonly used for freezing the specimen is liquid ethane; liquid nitrogen is used to maintain the temperature of the ethane near its melting point of -183 °C. With a freezing rate on the order of 1,000,000 K/sec, the fluid surrounding the specimen does not have time to form crystalline ice, which would damage the fragile sample; instead it is vitrified. Embedded within this layer of vitreous ice, the specimen is preserved in essentially its native state to near atomic resolution.  Using cryoEM on vitrified samples, investigators have visualized viruses, for example, at better than 0.4 nm resolution.

Cp3 incorporates many features that facilitate the process of preparing the vitrified specimen. The temperature of the liquid ethane can be held just above its melting point.  The removable liquid nitrogen workstation has a remote fill funnel and two removable covers. As liquid nitrogen is added to the workstation, a blanket of cold, dry nitrogen gas fills the workstation chamber, minimizing condensation of atmospheric oxygen and water vapor onto the surface of the ethane; it also generates a protective cryo interface for transferring the frozen hydrated grid within the workstation. The pneumatically assisted, multi-positional plunge piston fires the freshly blotted grid into the ethane pot at 1.7 m/sec. The workstation covers are positioned to allow easy access for removal and transport of the frozen hydrated specimen grid. One press of the quick disconnect pushbutton on the plunge rod allows the operator to quickly and easily disconnect the plunging tweezers and safely transfer the frozen hydrated grid to a pre-cooled cryo grid storage box located within the cryo grid box transfer pot. The frozen hydrated grids are fully protected as the liquid nitrogen filled transfer pot is removed from the workstation and transferred to a liquid nitrogen storage dewar or to the pre-cooled workstation of a cryo transfer holder for low electron dose imaging on the TEM.

Plunging with Cp3:  After the grid is plunged into the ethane pot (a) the plunge tweezers are disconnected from the plunge rod and, if desired, any excess ethane can be wicked off (b) using the pre-cooled filter paper that is held by a clip within the cryo workstation. Once the frozen hydrated grids are secure within the pre-cooled blue cryo grid box, the liquid nitrogen transfer pot (c) is removed to a liquid nitrogen transfer dewar (d) and the grid box can either be placed in a conical tube for storage under liquid nitrogen or transferred to the pre-cooled workstation of a cryo transfer holder for immediate viewing on the TEM.

The four images above are examples of the high quality of frozen hydrated preparations produced with Cryoplunge3.  (a) Image of several grid squares; TEM magnification 140X, ~ 0.01 e^-/Ǻ².  (b) Image of a single grid square; TEM magnification ~900X, electron dose 0.01 e^-/Ų.  (c) Higher magnification image of a portion of the grid square; 4700X, 0.1 e^-/Ų.  (d) Image from part of one hole; 59KX, 20 e^-/Ų.  Specimens were prepared on Quantifoil R1.2/1.3 macro machined holey carbon grids which were plasma cleaned using the Gatan Solarus 950 Advanced Plasma Cleaning System for 15 seconds at 50 Watts using the hydrogen/oxygen plasma.  All images were recorded on an FEI Tecnai F30 TEM with a Gatan 626 70°single tilt liquid nitrogen cryo transfer holder and a Gatan Ultrascan^® 4000.  Images courtesy of Dr. Chen Xu, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA. 

Cryoplunge3 is easy to use and provides clean, consistent results for the preparation of frozen hydrated specimens for cryoEM.  It incorporates many of the features found in more expensive plunge freezing instruments at a fraction of the cost while providing a versatile platform to facilitate a variety of specimen preparation protocols.

References

Dubochet, J., Groom, M. and Mueller-Neuteboom, S. (1982), Mounting of macromolecules for electron   microscopy with particular reference to surface phenomena and treatment of support films by glow discharge. Advances in optical and electron microscopy, Barrer, R. and Cosslett, V. E. (eds.), Academic Press, London, New York. 107-135.

Fukami A, Adachi K. (1965) A new method of preparation of a self-perforated micro plastic grid and its application.  J Electron Microscopy (Japan). 14(2):112-118.

Glaser, R, Downing, K, DeRosier, D, Chiu, W, Frank, J. (2007) Electron Crystallography of Biological Macromolecules.  Oxford University Press. 150-166.

Steinbrecht, RA, Zierold, K. (1987) Cryotechniques in biological electron microscopy. Berlin: Springer-Verlag. 47-54.

The author thanks Dr. David DeRosier of Brandeis University, Dr. David Nackashi of Protochips, Inc., and Mr. Dick Mitro, Dr. Steve Coyle and Dr. John Hunt of Gatan, Inc., for helpful comments.