Micromachining tools and correlative approaches for cellular cryo-electron tomography

Rigort, Alexander; Bäuerlein, Felix J.B.; Leis, Andrew; Gruska, Manuela; Hoffmann, Christian; Laugks, Tim; Böhm, Ulrike; Eibauer, Matthias; Gnaegi, Helmut; Baumeister, Wolfgang; Plitzko, Jürgen M.

doi:10.1016/j.jsb.2010.02.011

Cited by 235 publications

(232 citation statements)

References 41 publications

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“…S1B). The autogrid remains clamped in a cryoholder [cryoshuttle, (12)], which can be shuttered during transfers. A central mesh on the EM grid is selected and milling is performed under grazing angles by applying two milling patterns: One is located above the intended lamella region the second one below that region (compare Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In order to protect the vitrified specimen from frost contamination caused by ambient humidity and to ensure maximal thermal stability, we used a cryoshuttle together with a loading station as previously described (12). Both parts were adapted to a Polarprep 2000T cryosystem (Quorum) mounted on the FIB instrument.…”

Section: Methodsmentioning

confidence: 99%

“…Pilot studies have shown that cryoFIB milling can indeed be applied to frozen-hydrated material, rendering cellular samples transparent enough for TEM (11)(12)(13). However, the simple ablation geometries used in these studies would not permit to address structures embedded deeply in cellular volumes in a targeted manner.…”

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confidence: 99%

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Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

389

306

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“…Here, conventional resin-embedded samples are imaged by detecting the backscattered electron (BSE) signal from the block face. FIB-milling of frozen-hydrated specimen was applied without imaging the ultrastructure directly as a preparatory step for thinning samples to a suitable size for cryo-TEM or CET instead of cryosectioning (Hayles et al, 2010;Marko et al, 2007;Rigort et al, 2012;Rigort et al, 2010;Wang et al, 2012). Important contributions to high-resolution block-face imaging of frozen specimens in cryo-SEM were made by Paul Walter and colleagues showing that many subcellular structures are revealed in remarkable detail by special BSE detection after limited surface sublimation ('freeze-etching') and double-layer coating with platinum/carbon (Walther, 2008;Walther and Müller, 1999).…”

Section: Introductionmentioning

confidence: 99%

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Schertel

Snaidero

Hong-mei

et al. 2013

Journal of Structural Biology

170

113

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Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM)is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at −150°C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X=7.72 µm, Y=5.79 µm and Z=3.81 µm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a threedimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant Bacillus subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which can not be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.

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“…Recently several studies have demonstrated the feasibility of cryo-FIB milling to reduce the specimen thickness using small bacterial cells [6][7]. Here we report the technical advances in cryo-FIB processing of large mammalian cells, creating samples suitable for 3D structural analysis.…”

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confidence: 97%