Characterization of dentine structure in three dimensions using FIB-SEM

All biological tissues are three dimensional and contain structures that span a range of length scales from nanometres through to hundreds of millimetres. These are not ideally suited to current three-dimensional characterization techniques such as X-ray or transmission electron tomography. Such detailed morphological analysis is critical to understanding the structural features relevant to tissue function and designing therapeutic strategies intended to address structural deficiencies encountered in pathological states. We show that use of focused ion beam milling combined with scanning electron microscopy can provide three-dimensional information at nanometre resolution from biologically relevant volumes of material, in this case dentine.     

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.     

Method to deterministically study photonic nanostructures in different experimental instruments

We describe an experimental method to recover a single, deterministically fabricated nanostructure in various experimental instruments without the use of artificially fabricated markers, with the aim to study photonic structures. Therefore, a detailed map of the spatial surroundings of the nanostructure is made during the fabrication of the structure. These maps are made using a series of micrographs with successively decreasing magnifications. The graphs reveal intrinsic and characteristic geometric features that can subsequently be used in different setups to act as markers. As an illustration, we probe surface cavities with radii of 65 nm on a silica opal photonic crystal with various setups: a focused ion beam workstation; a scanning electron microscope (SEM); a wide field optical microscope and a confocal microscope. We use cross-correlation techniques to recover a small area imaged with the SEM in a large area photographed with the optical microscope, which provides a possible avenue to automatic searching. We show how both structural and optical reflectivity data can be obtained from one and the same nanostructure. Since our approach does not use artificial grids or markers, it is of particular interest for samples whose structure is not known a priori , like samples created solely by self-assembly. In addition, our method is not restricted to conducting samples.     

Imaging damage evolution in a small particle metal matrix composite

It is difficult to study effectively microstructural damage in metal matrix composites (MMCs) due to artefacts arising from traditional metallographic sample preparation techniques. The sectioning and imaging capabilities of the focused ion beam (FIB) microscope provide an excellent method for studying damage accumulation in MMCs.
The capabilities of the FIB system have been used to carry out a study of damage evolution in a powder-processed/hot-extruded Al2080/SiC_p MMC. Microvoid damage is found to be preserved accurately during FIB sectioning, allowing measurements of the fraction of decohered particles and the void area fraction. These microscopic damage measurements are correlated with the macroscopic damage parameter, D , as determined by density measurements.
Using transmission electron microscopy, the evolution of dislocation structures at the SiC–matrix interfaces has been examined. A previously unreported decohesion mechanism has been observed.     

Observation of human dentine by focused ion beam and energy-filtering transmission electron microscopy

Molar dentine was sliced into 100 nm ultrathin sections, by means of a focused ion beam, for observation by energy-filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10 nm wide and 50–100 nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

Nano-slit probes for near-field optical microscopy fabricated by focused ion beams

The near-field probes described in this paper are based on metallized non-contact atomic force microscope cantilevers made of silicon. For application in high-resolution near-field optical/infrared microscopy, we use aperture probes with the aperture being fabricated by focused ion beams. This technique allows us to create apertures of sub-wavelength dimensions with different geometries. In this paper we present the use of slit-shaped apertures which show a polarization-dependent transmission efficiency and a lateral resolution of < 100 nm at a wavelength of 1064 nm. As a test sample to characterize the near-field probes we investigated gold/palladium structures, deposited on an ultrathin chromium sublayer on a silicon wafer, in constant-height mode.     

Observation of the ion-mirror effect during microscopy of insulating materials

Utilizing the ion beam of a focused ion beam (FIB)/scanning electron microscope (SEM) microscope to investigate non‐conductive samples, we observe a mirror image very much similar to the one that is commonly obtained with the electron beam and the same samples. To our knowledge this is the first observation of what can be called ‘Ion‐Mirror Effect’. This effect is produced by a positive charging of the sample obtained by rastering with high‐energy ions (30 kV) and a subsequent imaging with low energy ones (5 kV). The proposed explanation is that first a positive charge is trapped within the sample and eventually the lower energy ions are deflected back by the latter, and hit the surface of the microscope chamber very similar to what happens in the ‘Electron‐Mirror Effect’. The mirror image is produced after detection of the electrons produced by the interaction between ions and the chamber materials.     

Microfabricated silicon dioxide cantilever with subwavelength aperture

We have developed a microfabricated SiO₂ cantilever with subwavelength aperture for scanning near-field optical microscopy (SNOM), to overcome the disadvantages of conventional optical fibre probes such as low reproducibility and low optical throughput. The microcantilever, which has a SiO₂ cantilever and an aperture tip near the end of the cantilever, is fabricated in a reproducible batch process. The circular aperture with a diameter of 100–150 nm is formed by a focused ion-beam technique. Incident light is directly focused on the aperture from the rear side of the cantilever using a focusing objective, and high optical throughput (10⁻² to 10⁻³) is obtained. The microcantilever can be operated as a SNOM probe in contact mode or in dynamic mode.