SCANNING VOL. 00, 1–10 (2012) C  Wiley Periodicals, Inc. The Application of STEM and In situ Controlled Dehydration to Bacterial Systems Using ESEM LECH STANIEWICZ 1 ,ATHENE M. DONALD 1 ,DEBBIE J. STOKES 2 ,NICHOLAS THOMSON 1 ,EASAN SIVANIAH 1 , ANDREW GRANT 3 ,DAVID BULMER 4 , AND ANJAM KHAN 4 1 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 2 FEI Company, Eindhoven, The Netherlands 3 Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, United Kingdom 4 Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle, United Kingdom Summary: Transmission imaging with an environ- mental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of elec- tron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little infor- mation available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and mi- croscopy employed by the authors for imaging bacte- ria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, al- though clearly in some sense artifactual, is thought to lead to structurally meaningful morphological infor- mation. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic- producing Cupriavidus necator along with wild-type and mreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown Contract grant sponsor: Medical Research Council; Contract grant numbers: G0801161, G0801212; Contract grant sponsor: Engi- neering and Physical Sciences Research Council; Contract grant num- bers: EP/P50385X/1, EP/P504120/1; Contract grant sponsor: FIE Company; Contract grant number: CASE studentship. Address for reprints: Lech Staniewicz, Cavendish Laboratory, Uni- versity of Cambridge, Cambridge, CB3 0HE, UK. E-mail: ltls2@cam.ac.uk Received 19 August 2011; Accepted with revision 19 October 2011 DOI 10.1002/sca.21000 Published online 00 XXXX 2012 in Wiley Online Library (wiley onlinelibrary.com) to exhibit clear internal differences between bacteria with and without plastic granules, while the mreC mutant of S. Typhimurium has an internal morphol- ogy distinct from that of the wild type. SCANNING 00: 1–10, 2012. C  2012 Wiley Periodicals, Inc. Key words: STEM, ESEM, bacteria, dehydration Introduction Light microscopy and transmission electron mi- croscopy (TEM) are two well-established techniques in the repertoire of the modern cell biologist. There is, however, a distinct gap between the two in terms of both image resolution and the complexity of the sample preparation process. By using an environmen- tal scanning electron microscope (ESEM) in combi- nation with a transmitted electron detector, a com- promise can be found between these two methods, permitting high-resolution images to be obtained of samples that have undergone minimal preparation. This technique, dubbed Wet STEM (scanning trans- mission electron microscopy), allows single cells to be imaged with strong contrast and with a resolution exceeding that accessible through conventional light microscopy. Wet STEM was first detailed in 2005, when Bogner’s group developed a method for combin- ing a Peltier temperature control stage and a detec- tor for the transmitted component of the electron beam (Bogner et al., 2005, 2007) to enable trans- mission imaging of material in any state of hydra- tion. While the alternative method of environmental capsules (such as those manufactured by Quantomix, Nes-Ziona, Israel) can be used to image wet material in a conventional high-vacuum SEM or TEM, this