Scanning electron microscopy
Since early 2020 the Geomicrobiology group has direct access to a new Zeiss Crossbeam 550L Cryo-FIB-SEM equipped with multiple detectors (Inlens SE, SESI, ESB, BSE, STEM), UniGIS System, XEI Evactron Zephyr Plasma Cleaner, EDS/EDX (UltimMax 100 / AZtecEnergy Advanced), Kleindiek Micromanipulator and Leica accessories for cryo sample preparation (Leica EM ACE600, EM VCT500, transfer shuttle and auto refilling device for EM VCT500).
Cryogenic focused ion beam scanning electron microscopy (Cryo-FIB-SEM)
This technique is used to image samples prepared using cryogenic fixation methods (e.g. plunge freezing and high pressure freezing) to maintain samples in as near-to-native as state as possible. By imaging samples at cryogenic temperatures, the oxidation state of oxidation sensitive samples will also be maintained. Through the application of a focused ion beam, gallium ions (Ga+) are used to remove material from the frozen block, one layer at a time. This enables serial block face imaging, resulting in the collection of a stack of images (Figure 1), which are then combined to produce 3D reconstructions of the region of interest.
Field emission scanning electron microscopy (FE-SEM)
The Crossbeam 550L is equipped with a field emission gun, allowing high resolution imaging of cell-mineral aggregates forming during microbial Fe cycling also in regular room temperature samples (not prepared by cryogenic methodologies). This includes the formation of Fe(III) minerals such as goethite by the nitrate dependent Fe(II)-oxidizing bacteria Acidovorax sp. BoFeN1 (Figure 2).
Transmission electron microscopy
Cryogenic transmission electron microscopy (Cryo-TEM)
Through external collaborations, the Geomicrobiology group has successfully applied cryo-TEM to study the influence of different substrate conditions on the development of intracellular cyctoplasmic membrane within phototropic Fe(II)-oxidizing bacteria Rhodopseudomonas palustris TIE-1 (Figure 3).
Bryce, C., Franz-Wachtel, M., Nalpas, N.C., Miot, J., Benzerara, K., Byrne, J.M., Kleindienst, S., Macek, B., Kappler, A. (2018). Proteome response of a metabolically flexible anoxygenic phototroph to Fe(II) oxidation. Applied and Environmental Microbiology, 84, e01166-18.
High resolution transmission electron microscopy (HR-TEM)
We use room temperature TEM to analyse magnetite nanoparticles produced by biogenic pathways such as Fe(III) reduction by bacteria such as Geobacter sulfurreducens. In a recent study, we exposed magnetite to Fe(II)-oxidizing or Fe(III)-reducing bacteria and used TEM (Figure 4) to image the materials before and after exposure to each type of organism. We were able to show that the organisms can use the magnetite without resulting in any morphological differences in the mineral structure, supporting the hypothesis that magnetite can behave as a biogeobattery in the environment.
Byrne, J.M., van der Laan, G., Figueroa, A.I., Qafoku, O., Wang, C., Pearce, C.I., Jackson, M., Feinberg, J., Rosso, K.M., Kappler, A. (2016). Size dependent microbial oxidation and reduction of magnetite nano- and micro-particles. Scientific Reports, 6, 30969.
Helium ion microscopy
He Ion Microscopy (HIM) is a relatively new technique in the field of high resolution microscopy in which helium ions (He+) are focused to form a primary beam, as opposed to electrons which are focused in conventional SEM. The helium ions can be used to form a smaller probe size with much smaller interaction volume at the sample surface compared to electrons. HIM has better material contrast and depth of focus compared to SEM. Furthermore, HIMs have charge compensation devices (flood gun) to enable imaging of non-conductive samples (such as organic material) without the requirement to sputter coat. HIM can thus provide unparalleled resolution and contrast compared to SEM. We recently used HIM to image the development of the twisted iron stalks, formed by microaerophilic Fe(II) oxidizing bacteria over a period of 4 weeks (Figure 5).
Byrne, J.M., Schmidt, M., Gauger, T., Bryce, C., Kappler, A. (2018). Imaging organic-mineral aggregates formed by Fe(II)-oxidizing bacteria using He-Ion Microscopy. Environmental Science and Technology Letters, 5, 209-213.