利用连续块面扫描电子显微镜（SBF-SEM）对人细胞有丝分裂保真度的三维超微结构可视化

Nuria   Ferrandiz; Stephen J. Royle

doi:10.21769/BioProtoc.4708

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Peer-reviewed

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM)

利用连续块面扫描电子显微镜（SBF-SEM）对人细胞有丝分裂保真度的三维超微结构可视化

NF Nuria Ferrandiz email

SR Stephen J. Royle

发布: 2023年07月05日第13卷第13期 DOI: 10.21769/BioProtoc.4708 浏览次数: 2147

评审: Nafisa M. JadavjiAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Cell Biology, featuring study using the protocol.

Apr 2022

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

Keywords: Chromosome missegregation (染色体错离)

Mitosis (有丝分裂)

Endoplasmic reticulum (内质网)

Volume EM (体积电镜)

Microscopy (显微镜)

Scanning electron microscopy (扫描电子显微镜)

Background

Cell division is essential for living organisms, as it is important for growth, repair, development, and reproduction. Accurate chromosome segregation during mitosis is essential to maintain genomic stability in cell division. Entry into mitosis involves a dramatic, large scale cellular reorganization. After the nuclear envelope breaks down, the chromosomes congress at the cell equator. The mitotic spindle, a complex subcellular machine, coordinates this congression and then the accurate segregation of sister chromatids to the two daughter cells. The combination of light and electron microscopy (EM), together with computer-based 3D reconstruction, shows that the mitotic spindle is localized in an exclusion zone, which is free of endomembranes: nuclear envelope and Golgi remnants, endoplasmic reticulum (ER), and vesicles (Puhka et al., 2007; Lu et al., 2009; Puhka et al., 2012; Champion et al., 2017; Nixon et al., 2017). Beyond the exclusion zone, the endomembranes are densely packed and mitochondria are randomly distributed. Recently, we described how chromosomes that do not congress to the cell equator can lie beyond the exclusion zone and become ensheathed by endomembranes, constituting a risk factor for chromosome instability (Ferrandiz et al., 2022). Our aim here is to provide a detailed step-by-step protocol to visualize, at the ultrastructural level, these chromosomes that become ensheathed by endomembranes. We use light microscopy to locate the cell of interest and then semi-automated large volume EM method (serial block-face scanning electron microscopy, SBF-EM) and 3D reconstruction to visualize the mitotic cell. The protocol will be useful for other applications where the total endomembrane content of mitotic cells, or indeed non-dividing cells, needs to be visualized.

Materials and reagents

Cell culture

DMEM/F-12 Ham (Merck Life Science, catalog number: D6421-6)
Fetal bovine serum (FBS) (Sigma, catalog number: F7524)
Trypsin-EDTA solution (Sigma, catalog number: T3924)
l-glutamine (Sigma, catalog number: G7513)
Penicillin/streptomycin (Gibco, catalog number: 15140-122)
Sodium bicarbonate (NaHCO₃) (Sigma, catalog number: D8662)
MatTek gridded glass-bottom culture dishes (MatTek Corporation, catalog number: P35G-1.5-14-CGRD)
Fugene HD transfection reagent (Promega, catalog number: E2312)
OptiMEM (Gibco, catalog number: 10149832)
GSK923295 (Selleckchem, catalog number: s7090)
Thymidine (Sigma, catalog number: T1895)
RO-3306 (Sigma-Aldrich, catalog number: SML0569)
SiR-DNA (Spirochrome, catalog number: SC007)
Poly-L-Lysine (Sigma-Aldrich, catalog number: P8920)
Concanavalin A (Sigma-Aldrich, catalog number: C2010)

Sample processing

Sodium phosphate dibasic (Na₂HPO₄) (Sigma, catalog number: S0876)
Sodium phosphate monobasic (Na HPO4) (Sigma, catalog number: S0751)
Potassium hydroxide (KOH) (Sigma, catalog number: 221473)
Glutaraldehyde 25% (Agar, catalog number: R1011)
Paraformaldehyde 16% (Thermo Fisher, catalog number: 28908)
Tannic acid, low molecular weight (Electronic Microscopy Science, catalog number: 21710)
OsO₄ (TABB, catalog number: O014)
Potassium ferrocyanide (Sigma, catalog number: 14459-95-1)
Thiocarbohydrazide (TABB, catalog number: T009)
Uranyl acetate (UA) (TAAB, catalog number: U008)
Methanol anhydrous (Sigma, catalog number: 322415)
Lead nitrate (TAAB, catalog number: L019)
l-Aspartic acid (Sigma, catalog number: A9256)
Ethanol molecular grade (Sigma, catalog number: 51976)
Agar 100 premix kit-Hard (Agar Scientific, catalog number: R1140)
Silver conductive paste (TAAB, catalog number: S384)
Gold/Palladium (80/20%) 60 mm × 0.1 mm (Quorum technologies, catalog number: SC500-314B)
0.1 M phosphate buffered (PB) saline (see Recipes)
Fixation buffer (see Recipes)
2% reduced osmium (see Recipes)
1% thiocarbohydrazide (TCH) solution (see Recipes)
1% uranyl acetate (UA) (see Recipes)
Lead aspartate solution (see Recipes)
Agar 100 premix kit (see Recipes)

Equipment

Nikon CSU-W1 spinning disc confocal system with SoRa upgrade (Yokogawa). Used with a Nikon, 20×/0.50 objective (Nikon) with optional 2.3× intermediate magnification or 60×, 1.40 NA, oil, Plan Apo VC objective (Nikon), and 95B Prime camera (Photometrics). The system has CSU-W1 (Yokogawa) spinning disk unit with 50 μm and SoRa disks (SoRa disk used), Nikon Perfect Focus autofocus, Okolab microscope incubator, Nikon motorized XY stage, and Nikon 200 μm z-piezo. Excitation was via 405, 488, 561, and 638 nm lasers with 405/488/561/640 nm dichroic and Blue, 446/60; Green, 525/50; Red, 600/52; FRed, 708/75 emission filters
Ultramicrotome (Leica Microsystems, EM UC7)
Gatan 3 View system installed on an FEI Quant 250 ESEM, with digital micrograph software (Gatan)

Software

NiS Elements (Nikon)
Digital micrograph software (Gatan)
Open-source programs for analyzing images such as:
1. Fiji (https://fiji.sc) (version 2.9.0)
2. Microscopy Image Browser (http://mib.helsinki.fi) (version 2.60)
3. IMOD (https://bio3d.colorado.edu/imod/) (version 4.10.49)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Ferrandiz, N. and Royle, S. J. (2023). 3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM). Bio-protocol 13(13): e4708. DOI: 10.21769/BioProtoc.4708.
Ferrandiz, N., Downie, L., Starling, G. P. and Royle, S. J. (2022). Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes. J Cell Biol 221(6): e202203021.