Precise and economic FIB/SEM for CLEM: with 2\xa0nm voxels through mitosis

Manja Luckner,Gerhard Wanner

doi:10.1007/s00418-018-1681-x

Abstract

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.

Highlights

Four ultrastructural techniques are established for 3D-reconstruction of biological specimens: (i) cryo-TEM tomography highest resolution, but limited in section thickness; (ii) serial block face sectioning (3View®; large volumes; limited resolution and charging problems; (iii) array tomography; and (iv) focused ion beam scanning electron microscopy (FIB/ SEM) tomography
As it is crucial to define the coordinates of a target area for re-localization in SEM we developed a variety of slides with coordinates, successively improved for different demands (Schroeder-Reiter et al 2012)
The entire EM preparation of cells was performed on customized laser marked coverslips/slides to track the position from light microscope (LM) through SEM investigation (Fig. 1)

Summary

Introduction

Four ultrastructural techniques are established for 3D-reconstruction of biological specimens: (i) cryo-TEM tomography highest resolution, but limited in section thickness (approx. 500 nm); (ii) serial block face sectioning (3View®; large volumes; limited resolution and charging problems; (iii) array tomography (non-destructive; limited resolution in z); and (iv) focused ion beam scanning electron microscopy (FIB/ SEM) tomography (larger volumes and highest resolution in z). 500 nm); (ii) serial block face sectioning (3View®; large volumes; limited resolution and charging problems; (iii) array tomography (non-destructive; limited resolution in z); and (iv) focused ion beam scanning electron microscopy (FIB/ SEM) tomography (larger volumes and highest resolution in z). There is no doubt, that cryo-TEM tomography is the state of the art technique for structural preservation and resolution of sub-cellular structures, with severe limitations, when investigating larger volumes in 3D. Comparing resolutions of 3View®, array tomography and FIB/SEM-tomography, there are no significant differences in xy As it is crucial to define the coordinates of a target area for re-localization in SEM we developed a variety of slides with coordinates, successively improved for different demands (Schroeder-Reiter et al 2012). Several modified protocols are available using thin embedding, but lack

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