MICROSCOPY RESEARCH AND TECHNIQUE 00:000–000 (2011) A Portable Cryo-Plunger for On-Site Intact Cryogenic Microscopy Sample Preparation in Natural Environments LUIS R. COMOLLI,1* ROBERT DUARTE,2 DENNIS BAUM,2 BIRGIT LUEF,1,3 1 1 1 3,4 KENNETH H. DOWNING, DAVID M. LARSON, ROSEANN CSENCSITS, AND JILLIAN F. BANFIELD 1Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 2Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 3Department of Earth and Planetary Science, Policy and Management, University of California, Berkeley, Berkeley, California 4Department of Environmental Science, Policy and Management, University of California, Berkeley, Berkeley, California KEY WORDS cryo-electron microscopy; cryo-plunging; environmental microbial communities; Archaea; extremophiles ABSTRACT We present a modern, light portable device specifically designed for environmental samples for cryogenic transmission-electron microscopy (cryo-TEM) by on-site cryo-plunging. The power of cryo-TEM comes from preparation of artifact-free samples. However, in many studies, the samples must be collected at remote field locations, and the time involved in transporting samples back to the laboratory for cryogenic preservation can lead to severe degradation artifacts. Thus, going back to the basics, we developed a simple mechanical device that is light and easy to transport on foot yet effective. With the system design presented here we are able to obtain cryo-samples of microbes and microbial communities not possible to culture, in their near-intact environmental conditions as well as in routine laboratory work, and in real time. This methodology thus enables us to bring the power of cryo-TEM to microbial ecology. Microsc. Res. Tech. 00:000–000, 2011. VC 2011 Wiley Periodicals, Inc. INTRODUCTION (Dubochet et al., 1982) and imaged cryo-sections of The first cryogenic transmission electron microscopy bacteria cultures (Dubochet et al., 1983). The first cryo- (cryo-TEM) grids (i.e., thin films of unsupported, vitreous TEM grid, essentially consisting of a thin film of an ice) were obtained 30 yr ago. Initially, the imaging tar- aqueous suspension spanning the free holes of a TEM gets were relatively large biological objects such as grid frozen as amorphous ice, was achieved by Adrian viruses and bacteria. Considerable expertise and crafts- and Dubochet (1984), and modern, routine cryo-TEM manship in cryo-grid preparation and technological pro- was thus started. gress in transmission electron microcopy (TEM) instru- While all fundamental issues underlying good pres- ments have extended the range of application of this ervation in the near-intact or ‘‘near-native’’ state are uniquely powerful technology to the study of biological conceptually the same from small biomolecules macromolecules with near atomic level resolution (Stahl- through biopolymers and intact bacterial cells and berg and Walz, 2008), organic compounds (Frederik and viruses, the demands across this range of samples can Sommerdijk, 2005) and near-intact bacteria (Milne and vary greatly. As recognized by Dubochet et al. (1985), Subramanian, 2009). A whole range of experimental the thin film of aqueous suspension vitrified in cryo- steps related to cryo-TEM sample preparation, data ac- TEM samples is self-stabilizing and the simplest cryo- quisition, and data processing have become highly stand- system designs gave the best results for suspensions of ardized and automated. Most of the techniques and auto- viruses and cells. The steps in preparing such samples mation in cryo-sample preparation for ‘‘Single Particle’’ are straight-forward: a sample droplet is placed on a cryo-TEM have been readily adopted in cryo-electron cryo-TEM grid (‘‘holey’’ or ‘‘lacey’’ carbon support), tomography (cryo-ET) of intact cells and viruses. mounted in tweezers; most of the liquid is blotted off; the grid is very quickly submerged into liquid ethane Obtaining vitreous ice with solutions and suspen- 8 sions ‘‘trapped’’ in a state nearly identical to the liquid at approximately its freezing point (90 K; the ethane phase, and without the artifacts of crystallization, was is held in a liquid nitrogen bath). Cryo-TEM grids are a goal unsuccessfully pursued for a long time. The designed to provide areas of unsupported vitreous ice; turning point was achieved by Bru¨ ggeller and Mayer these are formed by surface tension of the aqueous sus- (1980) when they realized the main obstacle had been pension on the grid during blotting. The method con- the size of the target bulk solution. Micrometer-scale liquid droplets could be frozen by spraying from a jet Additional Supporting Information may be found in the online version of this article. into amorphous, vitreous ice. Soon after, Dubochet and *Correspondence to: Luis R. Comolli, Life Sciences Division, Lawrence Berke- McDowall (1981) imaged vitreous droplets of pure ley National Laboratory, Berkeley, California. E-mail: [email protected] water by TEM. These initial ‘‘cryo-grids’’ were pre- Received 15 September 2011; accepted in revised form 11 November 2011 pared by spraying the water onto continuous thin car- Contract grant sponsor: Director, Office of Science, Office of Biological and Environmental Research, U.S. Department of Energy; Contract grant number: bon TEM grids with a nebulizer as they were ‘‘plunged’’ DEAC02-05CH11231 into ethane or propane at 808 K. They applied their DOI 10.1002/jemt.22001 technique to aqueous suspensions of biomolecules Published online in Wiley Online Library (wileyonlinelibrary.com). VC 2011 WILEY PERIODICALS, INC. 2 L.R. COMOLLI ET AL. Fig. 1. Natural environments rich in extremophile microbial com- numerous ponds and streams of AMD rich in biofilms. The goal is to munities. A: Shores of Lake Tyrrell, Victoria, Australia. This is a obtain cryogenic TEM samples directly on-site at these and similarly hypersaline lake and the pink color is due to photosynthetic bacteria. ‘‘out of ordinary’’ locations. [Color figure can be viewed in the online B: Entrance to the IMM, in Richmond, CA. The tunnels contain issue, which is available at wileyonlinelibrary.com.] sists of forming a thin layer of the suspension and cool- For these reasons, efforts were made to provide sys- ing it in the vitreous state under conditions such that tems for sample preparation (cryo-TEM grid blotting the sample remains in a given intact (or near-intact) and vitrification) with controlled humidity and temper- state. ature for the study of hydrated organic, biological, and To obtain the highest signal-to-noise from low dose colloidal dispersions. Controlled environmental vitrifi- cryo-TEM images, one sample-specific parameter to cation systems (CEVS; Bellare et al., 1988) have optimize is the thickness of the layer formed by the sus- become the standard for the study of cryo-TEM sam- pension. If the thickness approaches the largest dimen- ples and the basis for the ‘‘VitrobotTM’’ patented by the sion (diameter) of the target object, the result is an ori- University of Maastricht and licensed to FEI (Frederik entation constraint and forced surface interactions, pos- and Sommerdijk, 2005; Iancu et al., 2007). This and sibly causing sample degradation and thus should be similar devices such as the Gatan CP3 (Gatan, Pleas- avoided. In addition, surface effects at the air-water anton, CA) and the Leica EM GP (Leica Microsystems interface must be avoided. In the case of biological GmbH, Wetzlar, Germany) freezing systems are macromolecules and complexes with dimensions in the reported to produce consistent, reproducible, high qual- range of 5–20 nm (order of 10 nm), the vitrified sus- ity results. In theory, sample-specific protocols with pension should be only slightly thicker than the macro- computerized parameters allow new students and molecules dimensions, in general <50 nm. The very researchers to obtain the same consistent results at dif- large surface to volume ratio means that evaporation ferent times or places. can be critical, even on time-scales of milliseconds. In a This sophisticated automation of cryo-TEM sample typical thin film preparation under room conditions, preparation has significantly improved the ability to temperature 208C and humidity 40%, in one second study the most delicate nano-chemistry systems. How- the film looses 40 nm of thickness (Frederik and Storms, ever, the cryo-TEM field has lost some of the ability to 2005). In addition, the temperature of the sample would understand how to obtain optimal samples at the other change as a consequence of evaporation. Changes in the extreme: the coarser level of whole bacteria, microbial osmolarity can be in the order of 23 or more within 1– communities, and environmental samples. For whole 2sforfilmthicknessintheorderof100 nm. These bacteria samples, the vitreous ice surrounding the bac- dramatic changes in pH, sample and solute concentra- teria must be at least slightly thicker than the diame- tion can result in structural changes, associations, disso- ter of the bacteria (if they are not to be deformed). This ciations, and collapse of the structures. Nonetheless, means, in general, around and above 500 nm. The great progress in cryo-TEM was achieved utilizing cus- evaporation rate will only decrease the thickness by tom-made, ‘‘in-house’’ plunge freezing devices with man- 40 nm in 1 s, <10%. Issues such as solute concentra- ual sample blotting. However, this methodology requires tion and osmolarity, and change in temperature, are a high level of user dexterity (Iancu et al., 2007). This second order if the user is proficient. Equivalent user experience, including the perception of changes in samples can thus often be obtained using the simplest local climate to achieve robust artifact-free reproducibil- ‘‘in-house’’ or ‘‘home-made’’ cryo-plunger and the ity, is hard to acquire, transmit, and consistently repli- VitrobotTM. cate. Moreover, the use of cryo-TEM in the study of deli- There are many advantages in having a simple, cus- cate, vulnerable samples, such as weakly associated tom-made ‘‘in-house’’ cryo-plunger when dealing with macromolecules, micelles and lipid vesicles, and in nano- more complex microbial samples.