Dataset Information

Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa.

ABSTRACT: Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. This protocol was validated in: Curr Biol (2022), DOI: 10.1016/j.cub.2022.04.024.

SUBMITTER: Nicolas WJ

PROVIDER: S-EPMC9729855 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in <i>Allium cepa</i>.

Nicolas William J WJ Jensen Grant J GJ Meyerowitz Elliot M EM

Bio-protocol 20221205 23

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 ...[more]

PMID: 36561119

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Project description:Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following chemical fixation, staining, and mechanical sectioning, which limit attainable resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) offers the potential to image unfixed cellular samples at higher resolution while preserving their native structures, but it requires samples to be frozen free from crystalline ice and thin enough to image via transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate the native ultrastructure of unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue directly on cryo-EM grids via plunge-freezing, as opposed to high pressure freezing which is generally used for thick samples. Following vitrification, we use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid. In comparison to gallium FIB, which is commonly used for biological samples, xenon plasma FIB is powerful enough to efficiently mill large volume samples, such as human brain tissue. Additionally, our approach allows for lamellae to be generated at variable depth inside the tissue as opposed to being limited to starting at the surface of the tissue. Lamellae generated in Alzheimer's disease brain tissue and imaged by cryo-ET reveal intact subcellular structures including components of autophagy and potential tau fibrils. Furthermore, we visualize myelin revealing intact compact myelin and functional cytoplasmic expansions such as cytoplasmic channels and the inner tongue. From these images we also measure the dimensions of myelin membranes, providing insight into how myelin basic protein forces out oligodendrocyte cytoplasm to form compact myelin and tightly links intracellular polar head groups of the oligodendrocyte plasma membrane. This approach provides a first view of unfixed, never previously frozen human brain tissue prepared by cryo-plasma FIB milling and imaged at high resolution by cryo-ET.

Dataset Information

Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa.

Publications

Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in <i>Allium cepa</i>.

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