The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy

Hayles, M; Winter, D. A. Matthijs de; Schneijdenberg, Chris T.W.M.; Meeldijk, Johannes D.; Luecken, U; Persoon, Hans; Water, Jeroen de; Jong, Frank de; Humbel, Bruno M.; Verkleij, Arie J.

doi:10.1016/j.jsb.2010.07.004

Cited by 85 publications

(68 citation statements)

References 27 publications

(26 reference statements)

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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

171

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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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

171

113

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“…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.…”

mentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

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

395

306

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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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“…Crevasses can be somewhat reduced when cryosections are cut just 20 nm thick (Zhang et al 2004), but this severely reduces the cellular volume sampled in each tomogram. Recently, the cryo focused-ionbeam (FIB) milling method was developed as a way to produce cryosections with minimal ' cutting' artifacts (Hayles et al 2010 ;Marko et al 2007 ;Rigort et al 2010). Here, a beam of ions (typically Gallium) is used to thin a pellet of high-pressure-frozen cells.…”

Section: Better Specimensmentioning

confidence: 99%

Electron tomography of cells

Gan

Jensen

2011

Quart. Rev. Biophys.

148

130

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Abstract. The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution threedimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or ' tomogram '. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.

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The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy

Cited by 85 publications

References 27 publications

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

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

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Electron tomography of cells

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