A Comparison of Liquid Nitrogen and Liquid Helium As Cryogens for Electron Cryotomography
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Journal of Structural Biology 153 (2006) 231–240 www.elsevier.com/locate/yjsbi A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography Cristina V. Iancu, Elizabeth R. Wright, J. Bernard Heymann 1, Grant J. Jensen ¤ Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA Received 29 June 2005; received in revised form 16 November 2005; accepted 7 December 2005 Available online 4 January 2006 Abstract 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- deWned 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 »82 or »12 K, respectively, and the results were compared with particular interest in the doses (10–200 e¡/Å2) and resolutions (3–8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at »12 K through the Wrst several tens of e¡/Å2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no diVerence in the preservation of medium-resolution (3–5 nm) structural detail at the two temperatures. Tomo- graphic reconstructions produced with total doses between 10 and 350 e¡/Å2 showed better results at »82 K than »12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography. 2006 Elsevier Inc. All rights reserved. Keywords: Electron cryomicroscopy; Tomography; Helium cooling; Radiation damage; CryoEM 1. Introduction specimens to just a few kelvins with liquid helium, but early studies were highly variable and “unable to Wnd deWnite Biological materials can be imaged in transmission elec- evidence that there is an improvement in radiation resis- tron microscopes in a life-like, “frozen-hydrated” state tance on going from liquid nitrogen to liquid helium” through the use of specialized cryostages that keep samples (International Study Group, 1986). More recently, how- frozen while they are inside the microscope column. For ever, Stark et al. found that the so-called “cryoprotection these samples, radiation damage is the principal resolution factor” at 4 K (under liquid helium cooling) was 1.4 and 2.5 limitation, far exceeding others such as electron optical per- times better than at 98 K (under liquid nitrogen cooling) for formance. The Wrst cryostages cooled samples to »90 K 7 and 3 Å spacings, respectively, in two-dimensional protein through thermal contact with liquid nitrogen. After it was crystals (Stark et al., 1996). Part of the ambiguity in past observed that radiation damage proceeded much more work stemmed from the fact that previous microscopes slowly at low temperature, it was hoped that additional used either liquid nitrogen or liquid helium exclusively, so dose resilience might be realized through further cooling that comparisons had to be made between diVerent micro- (Glaeser, 1971). New microscopes were engineered to cool scopes in diVerent labs or settings. All previous reports have focused on the doses (»10 e¡/ Å2 or less) and resolutions (»7 Å or better) of interest to * Corresponding author. Fax: +1 626 395 5730. electron crystallography, and used the same basic measure- E-mail address: [email protected] (G.J. Jensen). ment—the fading of crystal diVraction patterns. We wish to 1 Present address: Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Insti- emphasize that the question of whether liquid helium cool- tutes of Health, Bethesda, MD 20892, USA. ing is actually advantageous or not depends, of course, on 1047-8477/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.12.004 232 C.V. Iancu et al. / Journal of Structural Biology 153 (2006) 231–240 the type of sample and goals of the project. For two-dimen- The temperature of the sample when liquid helium is pres- sional crystallographic studies probing near-atomic resolu- ent in the inner dewar was estimated by mounting a silicon tion, for instance, specimen atoms must remain in their diode thermometer on a special cartridge while the column original locations, and doses of just a few e¡/Å2 are typi- was open in the factory. For our microscope, that tempera- cally used. For single particle analysis studies seeking to ture was measured to be 11.5 K. During operation, a ther- resolve secondary structure, structures the size of helices mocouple provides real-time measurements of the and sheets must remain largely intact, and doses of »10– temperature of the “cryobox” immediately surrounding 20 e¡/Å2 are standard. Finally, for tomographic studies and in thermal contact with the cartridge. When the inner seeking to visualize shapes of individual proteins, radiolytic dewar is Wlled with liquid helium, this reads 10 K. When fragments of protein domains must remain only approxi- cooled with liquid nitrogen, it reads 82 K. mately in place, and doses of »20 to 200 e¡/Å2 are common. To begin our studies, simple dose series of several fro- Furthermore, while lower temperatures may or may not zen-hydrated biological samples were recorded at the two help reach any of these goals, potential disadvantages such temperatures, and the radiation damage trajectories were as increased charging or drift must also be considered. Thus observed. The Wrst sample tested was intact, frozen- successes or failures with liquid helium cooling in one con- hydrated cells of a small bacterium, Mesoplasma Xorum. text are not necessarily predictive in others. Cells were plunge frozen, inserted into the microscope, and Here, our focus is tomography. We compare liquid cooled to »82 K with liquid nitrogen. A suitable cell was nitrogen and liquid helium cooling with methods that are centered under the beam with less than 1 e¡/Å2 dose, and diVerent from all previous work in the nature of the sample, then approximately 70 exposures were recorded with 10 e¡/ the doses used, the type of data collected, the measures of Å2 each. Several key images are shown in the Wrst column quality, and the degree of control. Whole cells and individ- of Fig. 1. Next the grid was retracted into the liquid nitro- ual, large protein complexes are imaged through doses of gen cooled “multispecimen transfer system,” which resides 10–350 e¡/Å2. Image quality is measured both qualitatively within the column vacuum, and the liquid nitrogen in the and quantitatively in the resolution range of 3–6 nm. The inner dewar was replaced with liquid helium. After the tem- instrument used was one of the new series of “Polara” perature of the sample area settled to »12 K, the grid was microscopes from FEI, which allows liquid nitrogen and re-threaded onto the stage, allowed to cool (to »12 K), and helium to be exchanged repeatedly, all while a single grid is another Me. Xorum cell several microns away from the Wrst being imaged in a single microscope, without other con- was centered under the beam. An identical dose series was founding variables. Disappointingly, in this context we Wnd recorded (Fig. 1, second column). liquid helium cooling to be disadvantageous. In the com- At »82 K, the contrast from the cell membrane and mac- panion paper, we report several additional observations romolecular complexes inside the cell appeared qualita- that help explain this result (Wright et al., companion tively similar throughout the dose series, but did spread and paper). After conWrming that vitreous ice collapses from a smear gradually. In the Wrst image, the cell was punctuated low to a high density state when irradiated at »12 K, we go with small and distinct densities, which are the projections on to show that the collapse alone is not the immediate of internal macromolecules, and the membrane appeared as problem. Instead, we speculate that the key issue is how a dark, single band. In subsequent images, the internal den- radiolytic fragments aggregate within the high density ice. sities became less distinct and slightly rearranged. Never- theless, densities the size of single large protein complexes 2. Results could easily be traced through the images (circles in Fig. 1 column 1), suggesting that their basic shapes were pre- A 300 keV “G2 Polara” transmission electron micro- served, until small “bubbles” appeared after »160 e¡/Å2 scope from the FEI Company was installed in our lab at the and the internal structures were catastrophically perturbed. California Institute of Technology in the fall of 2003. It is At »12 K, the Wrst image was qualitatively indistinguish- equipped with a new cartridge-based sample holder speciW- able from the Wrst image at »82 K, but in subsequent cally designed to allow liquid helium cooling. An external images the membrane contrast faded quickly. The mem- dewar system is permanently mounted to the side of the brane became largely invisible after »40 e¡/Å2, and then column consisting of an inner “helium” dewar surrounded split into two dark bands separated by a light interior band. by an outer “nitrogen” dewar. The inner dewar is thermally This trilaminar structure had a slightly larger total width connected to the tip of the stage, so that when a cartridge is than the original membrane when it Wrst appeared (»80 e¡/ threaded onto the stage, it is cooled to near the temperature Å2), and then expanded non-uniformly as the dose series of the cryogen in the inner dewar within just a few minutes. continued. At the end (700 e¡/Å2), large bubbles appeared The Polara design is diVerent from some previous liquid between the dark outer layers (not shown).