The importance of the specimen support film for cryo TEM
By Linda Melanson, Life Science Applications Specialist, Gatan, Pleasanton, CA

An important factor that limits high resolution cryo TEM data collection is the preparation of the specimen. An important aspect of specimen preparation is the carbon support film to which the specimen is applied prior to plunge freezing in liquid ethane; surface charge, uniformity in thickness, planarity, and hole size and spacing all play a part in successful cryo preparations.

Holey carbon films, used with or without an additional overlay of thin (~ 5 nm) carbon, are a commonly used specimen support for cryo TEM studies of biological macromolecules. These holey support films can be made in the laboratory, or they can be purchased commercially. The preparation methods for most homemade holey supports involve casting a thin layer of resin such as polyvinyl formal (commercially known as Formvar® or Vinylec®), which is then further treated to produce the perforations. The perforated film is then applied to the surface of an electron microscope grid and a layer of carbon is evaporated onto the surface to enhance its stability and conductivity when exposed to the electron beam of the TEM. The resin layer is often removed by exposing it to solvent with the intent of leaving just the holey carbon layer. Beside the amount of time and the multiple steps required to prepare these support films, it is difficult to get reproducible results with regard to overall film thickness, hole size or hole spacing. Although somewhat expensive, commercially prepared films are an attractive alternative to homemade films because they are fabricated with a specified thickness, planarity, and hole shape, size and spacing all with minimal variation from one batch to the next.


SEM image (left) and optical LM image (right) 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.)

A common aspect of support films, whether homemade or commercially produced, is the variability of the surface charge. Although there are some exceptions such as certain membrane proteins, the majority of biological macromolecules prefer a hydrophilic surface. The visual indication of what occurs as the solution that contains the specimen is applied to the surface of the support film is an important qualitative measure of the surface charge. Ideally one wants this specimen solution, which is usually anywhere from 2 – 5 µl, to spread out evenly across the surface such that the majority of the specimen particles partition within the holes of the support without causing any changes to the macromolecular structure of the specimen. If the specimen solution ‘beads up’ when applied to the support film, the amount of specimen that actually partitions to the holes may be very small, thus limiting the amount of data that can be gathered.

Developing a reproducible means for preparing the surface charge of the support film prior to applying the specimen can improve the quality of the cryo preparation and maximize the amount of data that can be gathered from each specimen grid. To render specimen supports hydrophilic, plasma cleaning is now widely accepted as the method of choice. Commercial instruments designed for this purpose basically consist of a low vacuum chamber to contain the item to be cleaned and a means to introduce specific mixtures of gases that can be ionized by an electrical current to form the plasma. Absorption of electrical energy by the gas increases its temperature causing the ions to increase speed. These energetic ions can then interact with the exposed surfaces of the items being cleaned. Ion bombardment by an inert gas, such as argon, is similar to sandblasting; small amounts of material will be removed from the surface. When a gas mixture containing oxygen is used, bombardment of the surface causes chemical reactions that result in the removal of hydrocarbon contaminants from the surfaces of the items being cleaned. When cleaning hydrocarbon contamination from the surface of fragile holey carbon support films, it is important that the plasma is gentle enough such that it does not break the support during the cleaning process. As a rule of thumb, the specimen supports should be kept in a clean environment and be used within one hour following plasma cleaning.

Gatan’s Solarus™ (Model 950) Advanced Plasma Cleaning System. For more information please visit

http://www.gatan.com/specimenprep/solarus.php
and
http://www.gatan.com/knowhow
/knowhow13/index.htm#solarus

The Solarus™ Model 950 Advanced Plasma Cleaning System is a state-of-the-art, versatile and easy to use plasma cleaner. It provides optimal plasma power for any cleaning condition by incorporating a real-time radio frequency (RF) auto-matching network and a variable RF power supply operating at 10 to 50 watts. Mass flow controllers (MFC) for hydrogen, oxygen and argon gas automatically provide accurate gas flow to the cleaning chamber ensuring stable plasma production during the cleaning process. The unique H2/O2 gas mixture recipe (patent pending) provides superior cleaning with less sputter damage for even the most fragile specimens, and the plasma runs approximately 50% cooler than that from gas mixtures typically used such as Ar or Ar/O2. Using the interactive touch-screen interface, every 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. The spacious top entry specimen chamber can accommodate 40 to 50 specimen support films at one time or two TEM specimen holders, the latter of which can be inserted through ports at the front of the chamber. Process cycle rates of less than two minutes ensure high throughput performance.


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 milliliters 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. To prove the presence of oil contamination on the surface of the support film, two adjacent areas (a and b) were exposed to the electron beam for 20 and 60 seconds respectively. 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™ Advanced Plasma Cleaning System for 30 seconds using the exclusive H2/O2 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 of Gatan, Inc., Product Engineer for the Solarus™ Advanced Plasma Cleaning System.


Frozen hydrated, low dose images of 1-palmitoyl 1-oleoyl phosphatidylcholine (POPC) lipid vesicles with peptide prepared on a Quantifoil® R3.5/1 holey carbon specimen support. Quantifoil® was cleaned in the Solarus™ Advanced Plasma Cleaning System for 30 seconds using the exclusive H2/O2 gas mixture. Three microliters of the lipid/peptide solution was applied to the surface of the holey support followed by blotting and plunge freezing into liquid ethane at -172°C using the Gatan CryoPlunge. Images were recorded on a JEM2100 at 200kV with a Gatan US4000 CCD camera. (a) Survey image showing distribution of the peptide decorated lipid vesicles (total electron dose approximately 2 e-/Å2.) (b and c) Higher magnification images at 15kX and 60kX (total electron dose 7 and 15 e-/Å2 respectively) with defocus setting of 1.5 µ. Scale bars: (a) 1.5µ, (b) 400nm and (c) 100nm. Images courtesy of Mr. Htet Khant, National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, TX. USA.



Using a reproducible method for cleaning holey carbon support films is important because it improves the even spreading of the specimen solution over the surface of the support. With its proprietary H2/O2 gas mixture recipe, the Solarus™ Advanced Plasma Cleaning System can improve the surface characteristics for holey carbon support films by gently and efficiently removing hydrocarbon contamination, resulting in more consistent and more uniform specimen distribution in cryo preparations and therefore more productive data collection sessions at the electron microscope.

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.

Harris, JR. (1997). Negative Staining and Cryoelectron Microscopy. Oxford: BIOS Scientific Publishers Limited. 12-21.

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

The author thanks Dr. David DeRosier and Dr. Daniela Nicastro of Brandeis University, Dr. David Nackashi of Protochips, Inc., Dr. Ken Sautter of Yield Engineering Systems and Mr. Dick Mitro of Gatan, Inc., for helpful comments.

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