Elsevier

Micron

Volume 62, July 2014, Pages 66-78
Micron

Review
Serial sectioning methods for 3D investigations in materials science

https://doi.org/10.1016/j.micron.2014.03.002Get rights and content

Highlights

  • Serial sectioning methods SBEM, 3D AFM and 3D FIB for 3D reconstruction are compared.

  • Suggestions for the choice of optimum parameters are listed.

  • Applications for each technique are provided.

  • Examples of SBEM and 3D FIB combined with EDS are presented.

  • The impact of these methods on materials science is outlined.

Abstract

A variety of methods for the investigation and 3D representation of the inner structure of materials has been developed. In this paper, techniques based on slice and view using scanning microscopy for imaging are presented and compared. Three different methods of serial sectioning combined with either scanning electron or scanning ion microscopy or atomic force microscopy (AFM) were placed under scrutiny: serial block-face scanning electron microscopy, which facilitates an ultramicrotome built into the chamber of a variable pressure scanning electron microscope; three-dimensional (3D) AFM, which combines an (cryo-) ultramicrotome with an atomic force microscope, and 3D FIB, which delivers results by slicing with a focused ion beam. These three methods complement one another in many respects, e.g., in the type of materials that can be investigated, the resolution that can be obtained and the information that can be extracted from 3D reconstructions. A detailed review is given about preparation, the slice and view process itself, and the limitations of the methods and possible artifacts. Applications for each technique are also provided.

Introduction

Microscopy classically delivers only two-dimensional (2D) images, without providing direct quantitative information about the three-dimensional (3D) internal structure of a material. However, in the life sciences and materials science this knowledge is extremely important, and has fostered interest in 3D reconstructions of all types of materials. The 3D morphology of biological objects determines their functionality (Denk and Horstmann, 2004). The properties of materials are strongly dependent on their design, including micro- and nanostructures. There are numerous examples of steric features such as anisotropic grains, precipitates, intergranular phases, crack distributions in deformation zones (Möbus and Inkson, 2007), for which a 3D analysis of the structure of the material is fundamental.

There were several attempts early on in the history of microscopy to gather 3D models of microstructures. In 1876, Born published a 3D reconstruction of anatomical parts of amphibians based on serial sectioning combined with light microscopy (Born, 1876). Since that time, various microscopic techniques have been developed for both sectioning and imaging, additionally featuring an increase in microscopic resolution. The reader is referred to review articles such as (Midgley et al., 2007) concerning 3D methods by angular tomography. The discussion here will be restricted to 3D investigations using serial sectioning methods in combination with scanning microscopy techniques such as serial block-face scanning electron microscopy (SBEM), 3D atomic force microscopy (3D AFM) at room and cryogenic temperature and 3D focused ion beam (3D FIB).

Section snippets

Methods used for serial sectioning

Serial sectioning at the micro/nanoscale can be performed by microtomy, ion milling or consecutive polishing steps.

Microtomy is an established sample preparation method that was initially invented for light and transmission electron microscopic applications, but is now also used for preparing specimens for scanning electron microscopy (SEM), ion microscopy with an FIB, and AFM. However, ultramicrotomy is restricted to soft materials and metals ductile enough to be cut by a diamond knife. Still,

Serial block-face scanning electron microscopy (SBEM)

This method was developed by Denk and published in a landmark paper in 2004 (Denk and Horstmann, 2004). It enables the reconstruction of the internal structures of soft materials over hundreds of microns providing 3D information with an imaging resolution typical for SEM. Thus, the gap between high-resolution tomography in a transmission electron microscope (TEM) and light microscopy is bridged. A prerequisite for this method is an electron microscope that enables imaging of electrically

3D AFM

Another method for 3D reconstruction using serial sectioning is AFM combined with ultramicrotomy. The method can be applied for serial section tomography of typically biological and polymeric materials. There are two basic instrumentation setups that perform AFM 3D reconstruction both at RT and at cryo conditions. The RT instrument is automated and allows an acquisition of up to 10 sections per hour. The block face is imaged using a scanning probe microscope (SPM) NTEGRA (NT-MDT, Moscow,

3D FIB

The previous two instruments are typically applicable for soft materials and specimens that are sufficiently ductile that they can be cut with a diamond knife. For most other materials, a dual beam-focused ion beam microscope (DB-FIB), which combines an SEM and a precisely focused ion beam, may be the best choice. With the ion beam milling of hard materials, serial sectioning can thus be performed. The advent of DB-FIB systems compared to a single focused ion beam instrument (Inkson et al., 2001

Discussion and comparison of the methods SBEM, 3D AFM and 3D FIB

As shown in Table 1, the three methods presented here differ especially in lateral resolution and sample volume that can be processed. SBEM and 3D AFM are very similar as far as serial sectioning (cutting with a diamond knife) is concerned. How serial sectioning is performed determines the possible slice thickness, which in both cases can be quoted as greater 10 nm. Concerning SBEM Mancuso et al. (2010) used a slice thickness of 10 nm and Thompson et al. (2013) 15 nm. In Alekseev et al., 2009,

Conclusions

With the methods SBEM, 3D AFM and 3D FIB a 3D representation of the inner morphology of a material can be gained with high resolution. In images recorded of the block face, the resolution is limited by resolution of the microscope. In the direction perpendicular to the block face, the resolution is limited by minimum slice thickness and generally is lower than the resolution in the image plane. This may have to be taken into account if a material is not isotropic, but its inner structures have

Acknowledgements

The authors would like to thank Prof. Dr. Ferdinand Hofer and Dr. Nadejda B. Matsko for fruitful suggestions. We are grateful to Manuel Paller for generating the schematics of SBEM and FIB. This work was financially supported by Austrian Cooperative Research (ACR), Vienna.

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