Protocol for high-throughput electron tomography exemplified on centrioles in primary human plasma cells

Sebastian Köhrer; Tobias Dittrich; Martin Schorb; Isabella Haberbosch; Yannick Schwab; Alwin Krämer

doi:10.1016/j.xpro.2023.102373

Protocol for high-throughput electron tomography exemplified on centrioles in primary human plasma cells

STAR Protoc. 2023 Sep 15;4(3):102373. doi: 10.1016/j.xpro.2023.102373. Epub 2023 Jun 22.

Authors

Sebastian Köhrer¹, Tobias Dittrich², Martin Schorb³, Isabella Haberbosch⁴, Yannick Schwab⁵, Alwin Krämer⁶

Affiliations

¹ Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ), and Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany. Electronic address: sebastian.koehrer@embl.de.
² Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ), and Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany; Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany; Amyloidosis Center, University of Heidelberg, 69120 Heidelberg, Germany.
³ Electron Microscopy Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. Electronic address: martin.schorb@embl.de.
⁴ Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ), and Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany; Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany.
⁵ Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany; Electron Microscopy Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
⁶ Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ), and Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany; Department of Internal Medicine V, University of Heidelberg, 69120 Heidelberg, Germany. Electronic address: a.kraemer@dkfz-heidelberg.de.

Abstract

Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138^pos plasma cells. The protocol comprises steps for electron microscopy sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining. For complete details on the use and execution of this protocol, please refer to Dittrich et al.¹.

Keywords: High Throughput Screening; Microscopy.

MeSH terms

Centrioles*
Electron Microscope Tomography*
Humans
Microscopy, Electron, Transmission
Plasma Cells
Specimen Handling