ReviewSerial sectioning methods for 3D investigations in materials science
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.
References (77)
- et al.
Three-dimensional chemical analysis of laser-welded NiTi–stainless steel wires using a dual-beam FIB
Acta Mater.
(2013) Foundations of environmental scanning electron microscopy
(1988)- et al.
Radiation damage in the TEM and SEM
Micron
(2004) - et al.
The application of ultramicrotomy to the electronoptical examination of surface films on aluminium
Corros. Sci.
(1978) - et al.
Stochastic 3D modeling of La0.6Sr0.4CoO 3-δ cathodes based on structural segmentation of FIB-SEM images
Comput. Mater. Sci.
(2013) - et al.
Computational methods and challenges for large-scale circuit mapping
Curr. Opin. Neurobiol.
(2012) - et al.
3D determination of grain shape in a FeAl-based nanocomposite by 3D FIB tomography
Scripta Mater.
(2001) - et al.
Investigation of slice thickness and shape milled by a focused ion beam for three-dimensional reconstruction of microstructures
Ultramicroscopy
(2014) - et al.
Axon tracking in serial block-face scanning electron microscopy
Med. Image Anal.
(2009) - et al.
Modeling of flow in a polymeric chromatographic monolith
J. Chromatogr. A
(2011)
Investigation of orientation gradients around a hard Laves particle in a warm-rolled Fe3Al-based alloy using a 3D EBSD-FIB technique
Acta Mater.
Reconstruction and visualization of complex 3D pore morphologies in a high-pressure die-cast magnesium alloy
Mater. Sci. Eng. A
Image-based modeling of the response of experimental 3D microstructures to mechanical loading
Scripta Mater.
Contour-propagation algorithms for semi-automated reconstruction of neural processes
J. Neurosci. Methods
Nanoscale tomography in materials science
Mater. Today
The application of focused ion beam microscopy in the material sciences
Mater. Charact.
Quantitative characterization of microfiltration membranes by 3D reconstruction
J. Membr. Sci.
Automated three-dimensional X-ray analysis using a dual-beam FIB
Ultramicroscopy
Three-dimensional analysis of microstructures in titanium
Acta Mater.
Automated serial sectioning for 3-D analysis of microstructures
Scripta Mater.
3-D tomography by automated in situ block face ultramicrotome imaging using an FEG-SEM to study complex corrosion protective paint coatings
Corros. Sci.
3D microstructural characterization of nickel supperalloys via serial-sectioning using a dual beam FIB-SEM
Scripta Mater.
Influence of the strongly anisotropic cross-section morphology of a novel polyethersulfone microfiltration membrane on filtration performance
Sep. Purif. Technol.
Techniques for generating 3-D EBSD microstructures by FIB tomography
Mater. Charact.
Three-dimensional distribution, morphology, and nucleation site of intragranular ferrite formed in association with inclusions
Mater. Sci. Eng.
Quantification of the pore size distribution (porosity profiles) in microfiltration membranes by SEM, TEM and computer image analysis
J. Membr. Sci.
Three-dimensional electrical property mapping with nanometer resolution
Adv. Mater.
Local organization of graphene network inside graphene/polymer composites
Adv. Funct. Mater.
Three-dimensional textural and chemical characterization of polyphase inclusions in spodumene using a dual focused ion beam – scanning electron microscope (FIB-SEM)
Can. Mineral.
A direct and quantitative image of the internal nanostructure of nonordered porous monolithic carbon using FIB nanotomography
J. Microsc.
High-resolution three-dimensional reconstruction: a combined scanning electron microscope and focused ion-beam approach. J
Vac. Sci. Technol. B
Ueber die Nasenhöhlen und den Thränennasengang der Amphibien
Morph. Jb
Wiring specificity in the direction-selectivity circuit of the retina
Nature
Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure
PLoS Biol.
CASINO V2.42: a fast and easy-to-use modeling tool for scanning electron microscopy and microanalysis users
Scanning
Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples
J. Microsc.
Analysis of native structures of soft materials by cryo scanning probe tomography
Soft Matter
Scanning Electron Microscopy and X-ray Microanalysis
Cited by (79)
Numerical modelling of the mesofracture process of sintered 316L steel under tension using microtomography
2021, Engineering Fracture MechanicsImage-based numerical modeling of the tensile deformation behavior and mechanical properties of additive manufactured Ti–6Al–4V diamond lattice structures
2021, Materials Science and Engineering: AA Data-Driven Approach to Generating Stochastic Mesoscale 3D Shale Volume Elements From 2D SEM Images and Predicting the Equivalent Modulus
2023, International Journal of Applied MechanicsThe ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri
2023, Cell Biology International