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Application Note
IN SITU IMAGING OF BACTERIUM-HOST
INTERACTIONS IN 3D
related instruments: EM VCT100 / EM VCT500
2
IN SITU IMAGING OF BACTERIUM-HOST
INTERACTIONS IN 3D
Application Note for Leica Pre-Tilted Holder for AutoGrids
João M. Medeiros, Désirée Böck, Martin Pilhofer, Dr.
Institute of Molecular Biology & Biophysics, Eidgenössische Technische Hochschule Zürich, CH-8093 Zürich, Switzerland
In order to better understand bacterial pathogenicity or symbiosis, it is necessary to investigate the bacterium-host interactions that are
essential for these processes. Electron cryo-tomography is capable of resolving these events in high-resolution 3D reconstructions, albeit
solely for thin samples. Cryo-focused ion beam milling effectively lifts this restriction by providing an artifact-free thinning method that can
be applied to most biological samples, such as bacteria-infected eukaryotic cells.
The Leica Pre-Tilted Holder for AutoGrids (Fig.1) was crucial for routinely studying cellular interactions in a near-native state, as it synergi-
zes with the Leica loading box, VCT, high-vacuum sputter coater and cryo-stage, in order to provide a robust and reproducible workow.
An overview on using cryo-focused ion beam milling and electron cryo-tomography to study intracellular bacteria in their in vivo niche can
be found in: Medeiros, J. M., Böck, D., & Pilhofer, M. (2017). Imaging bacteria inside their host by cryo-focused ion beam milling and elec-
tron cryotomography. Current Opinion in Microbiology, 43, 62–68. http://doi.org/10.1016/j.mib.2017.12.006.
Workow
The experiment starts by growing the required cells in their respective culture medium and conditions. After infection, the cells are depo-
sited onto electron microscopy grids, the excess liquid is blotted and plunge frozen in an ethane-propane mixture cooled by liquid nitrogen.
The grids can be stored under liquid nitrogen until further use.
Prior to the sample thinning step, the grids are secured in a stabilizing support. They are then loaded onto the Leica Pre-Tilted Holder for Au-
toGrids in the Leica loading box. This holder is retrieved by the Leica VCT, which is then pumped to high vacuum and used to ferry the holder
onto the focused ion beam microscope. Here, a scanning electron column is used to image the grid surface and identify potential targets for
milling. The targets are ablated in two parallel rectangular patterns, leading to formation of a lamella with the remainder of the biological
material (Fig. 2). This lamella is thin enough for electron cryotomography studies (Fig. 3). When all targets are milled, the holder is retrieved
from the microscope by the Leica VCT and taken to the loading box where the grids are removed and stored until the transmission electron
microscope is available for tomography.
Fig. 1: Leica Pre-Tilted Holder for Autogrids
LNT Application Note - IN SITU IMAGING OF BACTERIUM-HOST INTERACTIONS IN 3D 3
Fig. 2: Scanning electron microscope (SEM) and focused ion beam (FIB) perspectives during the milling procedure. The area mar-
ked by the green squares is ablated with the ion beam to generate a section of material thin enough for imaging in the transmission electron
microscope, the lamella. Scale bars: 10 µm.
Fig. 3: Tomographic slice of a bacteria infected amoeba cell. Electron cryp-tomography can capture 3D snapshots of the native environ-
ment of complex relationships, such as pathogenicity or symbiosis.
© 2018 by Leica Microsystems GmbH.
Subject to modications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.
Leica Mikrosysteme GmbH | Vienna, Austria
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Related instruments:
EM VCT100 / EM VCT500
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