Multiscan ccd camera

Transmission electron microscopy (TEM) was performed according to^{49 (link)}. Briefly, the bacteria were fixed with 2% glutaraldehyde in cacodylate buffer (75 mM, pH 7.0) for 2 h, postfixed with 1% osmium tetroxide at 4 °C overnight. The samples were dehydrated through a series of graded acetone concentrations (30–100%) and finally embedded in plastic according to^{50 (link)}. Ultrathin sections were obtained with a ultramicrotome (Ultracut E, Leica-Reichert-Jung, Nußloch, Germany) and stained with uranyl acetate followed by lead citrate^{51 (link)}. Sections were viewed with a LEO 906 E TEM (LEO, Oberkochen, Germany) operated at 100 kV and equipped with the MultiScan CCD Camera (Model 794) of Gatan (Munich, Germany) using the Digital Micrograph software version 2.0.2 from Gatan to acquire, visualize, analyze, and process image data.

Cryo-tomographic tilt series were acquired on a Tecnai Polara transmission electron microscope (FEI Company Inc., Hillsboro, OR, USA) operated at 300 keV, equipped with a post-column GIF2002 energy filter and a slow scan 2048 × 2048 pixel Multiscan CCD camera (Gatan Inc., Pleasanton, CA, USA). Images were recorded in zero-loss mode (energy filter slit width: 20 eV) under low-dose conditions at a final magnification of 64 171, resulting in a pixel size of 4.72 Å on the object level. Tilt series taken at an intended underfocus of −8 μm comprised projections from −65° to 65° with an angular increment of 1.5°. For data acquisition, the Xplore3D software (FEI Company Inc.) was used. Projection alignment using at least eight gold markers and tomographic reconstruction by weighted back projection was done using the procedures implemented in the TOM Toolbox [57 (link)].

For the identification of external appendages by TEM, samples were deposited as whole mounts on 400 mesh carbon-coated copper grids (Quantifoil Micro Tools, GmbH Jena). The grids were imaged within 1 h with a Philips/FEI CM12 LaB₆ microscope at 100 kV onto a Gatan 1024 × 1024 pixel MultiScan CCD camera. Images were recorded and processed using Gatan Digital Micrograph software. On the order of 20 fields of view were observed for fermenting, Fe(III) citrate reducing and HFO reducing cell samples (20, 35, and 25, respectively).
Whole mounts of the products of Fe(III)-citrate and HFO reduction, deposited on carbon-coated gold or copper grids (Quantifoil Micro Tools, GmbH Jena), were imaged with a FEI CM300UT FEG-UT microscope. Selected area electron diffraction (SAED) of micron-scale areas and Fourier Transforms of high-resolution transmission electron microscopy (HR-TEM) images were used for phase identification with the Java Electron Microscopy Software (JEMS) (Stadelmann, 2012 ); the structural data of iron minerals was taken from the Inorganic Crystal Structure Database (ICSD) (ICSD, FIZ Karlsruhe, Germany and NIST, U.S. Department of Commerce, 2012).

The crystalline nature and lattice parameters were determined by X-ray diffraction (XRD, Rigaku Ultima IV X-ray diffractometer, Tokyo, Japan) using Cu Kα radiation (λ = 0.154 nm). The data were analyzed in the 2θ range with a scanning step of 2^o per minute. Using Fourier transform infrared spectrometer (FTIR, Perkin Elmer spectrum RX1 spectrometer, Chennai, India), the infrared spectrum was recorded in the wavelength ranging from 400 to 4000 cm⁻¹ using the KBr pellets technique. The morphology and particle size were characterized using a high-resolution transmission electron microscope (HRTEM) (JEOL-JEM-3010, JEOL USA Inc, MA, USA) operating at an accelerating voltage of 200 kV and at a magnification range of 40,000×–50,000×. The particles were deposited on a 300-mesh carbon-coated copper grid. A Gatan 794 multiscan CCD camera was used for image acquisition and the particle size was analyzed using ImageJ software.

The morphology and structure of VLPs from crude supernatants was assessed with two different electron microscopy techniques, including negative staining transmission electron microscopy (NS-TEM), and cryogenic transmission electron microscopy (cryo-TEM) as described previously [22 (link)]. For NS-TEM, 8 µL of sample were deposited on carbon-coated cooper grids (Micro to Nano, Wateringweg, The Netherlands) previously subjected to a glow discharge treatment (PELCO, Fresno, CA, USA) and incubated at room temperature for 1 min. Excess sample was carefully drained off the grid with the aid of filter paper. Then, 8 µL of 2% w/v uranyl acetate were added, and samples were incubated for 1 min at room temperature. Excess stained was drained off and grids were dried at room temperature until analysis. Sample visualization was performed in a JEM-1400 transmission electron microscope (JEOL, Akishima, Tokyo, Japan) equipped with an ES1000W Erlangshen charge-coupled device camera (model no. 785, Gatan, Pleasanton, CA, USA).
For cryo-TEM, 2 µL of sample were blotted on 400 mesh glow discharged Holey carbon grids (Micro to Nano). Samples were then plunged into liquid ethane at −180 °C using a Leica EM GP cryogenic workstation (Leica Microsystems) and visualized in a JEM-2011 electron microscope (JEOL) operating at 200 kV and equipped with a CCD multiscan camera (model no. 895, Gatan).