A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

Mahamid, Julia; Schampers, Ruud; Persoon, Hans; Hyman, Anthony A.; Baumeister, Wolfgang; Plitzko, Jürgen M.

doi:10.1016/j.jsb.2015.07.012

Cited by 138 publications

(88 citation statements)

References 44 publications

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“…If the samples are too thick for electron beam to penetrate, ultrathin sectioning under LN2 environment needs to be executed before the sample can be loaded into the cryo-TEM. Sometimes high-pressure freezing should be performed instead of fast frozen for large cells or tissues, to get vitreous ice for the whole volume (Mahamid et al 2015).…”

Section: Protocol Discussionmentioning

confidence: 99%

The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

Lei

Wang

2018

Biophys Rep

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Correlative light and electron microscopy is a powerful technique for identification and determination of the structures of interested macromolecules in situ. Combined with sample vitrification, it would be much easier to preserve the native state of macromolecule complexes and distinguish them from the crowded structure environment. In this article, we present a detailed process for the application of the CorrSight system, a light microscope equipped with a cryo module, in combination with a cryo-electron microscope. A relatively long course of up to 7-8 h for cryo module preparation and multichannel light microscopy imaging of vitrified specimen can be sustained. Correlation of light and electron microscopy images at both grid levels to locate squares and square level to locate target particles, and verification of target particles can be performed with the help of AutoEMation software. Cryo-electron tomography is used for obtaining the three-dimensional structure information.

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Section: Protocol Discussionmentioning

confidence: 99%

The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

Lei

Wang

2018

Biophys Rep

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“…The recent development of a Volta-potential phase plate enabled researchers to overcome this problem [14,15] (Figure 2b). Indeed, future applications will greatly benefit from cryo-ET setups that combine FIB milling, the use of direct electron detectors and Volta phase plates [16], as highlighted by studies of chloroplasts and Golgi ultrastructures in vitreous Chlamydomonas cells [17,18], S26 proteasomes in intact hippocampal neurons [19], organelle organization in C. elegans embryos and adult worms [20,21] and of the translocon-associated protein complex (TRAP) at the ER of human fibroblasts [22].…”

Section: Cellular Cryo-etmentioning

confidence: 99%

Structural Biology outside the box — inside the cell

Plitzko

Schuler

Selenko³

2017

Current Opinion in Structural Biology

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Recent developments in cellular cryo-electron tomography, in-cell single-molecule Förster resonance energy transfer-spectroscopy, nuclear magnetic resonance-spectroscopy and electron paramagnetic resonance-spectroscopy delivered unprecedented insights into the inner workings of cells. Here, we review complementary aspects of these methods and provide an outlook toward joint applications in the future.

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“…Initially developed as an alternative to cryo-sectioning, cryo-focused ion-beam (FIB) milling has been recently shown to be the best method so far to obtain thin slices of larger samples [32]. In this method, a focused ion-beam cuts out a thin lamella with high precision.…”

Section: Bridging the Gap Between Cells And Atomsmentioning

confidence: 99%

Electron cryomicroscopy as a powerful tool in biomedical research

Quentin

Raunser

2018

J Mol Med

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A human cell is a precisely regulated system that relies on the complex interaction of molecules. Structural insights into the cellular machinery at the atomic level allow us to understand the underlying regulatory mechanism and provide us with a roadmap for the development of novel drugs to fight diseases. Facilitated by recent technological breakthroughs, the Nobel prize-winning technique electron cryomicroscopy (cryo-EM) has become a versatile and extremely powerful tool to solve routinely near-atomic resolution three-dimensional protein structures. Consequently, it has become the focus of attention for structure-based drug design. In this review, we describe the basics of cryo-EM and highlight its growing role in biomedical research. Furthermore, we discuss latest developments as well as future perspectives.

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A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

Cited by 138 publications

References 44 publications

The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

Structural Biology outside the box — inside the cell

Electron cryomicroscopy as a powerful tool in biomedical research

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