Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

Argon broad ion beam tomography in a cryogenic scanning electron microscope: a novel tool for the investigation of representative microstructures in sedimentary rocks containing pore fluid

The contribution describes the implementation of a broad ion beam (BIB) polisher into a scanning electron microscope (SEM) functioning at cryogenic temperature (cryo). The whole system (BIB‐cryo‐SEM) provides a first generation of a novel multibeam electron microscope that combines broad ion beam with cryogenic facilities in a conventional SEM to produce large, high‐quality cross‐sections (up to 2 mm²) at cryogenic temperature to be imaged at the state‐of‐the‐art SEM resolution. Cryogenic method allows detecting fluids in their natural environment and preserves samples against desiccation and dehydration, which may damage natural microstructures. The investigation of microstructures in the third dimension is enabled by serial cross‐sectioning, providing broad ion beam tomography with slices down to 350 nm thick. The functionalities of the BIB‐cryo‐SEM are demonstrated by the investigation of rock salts (synthetic coarse‐grained sodium chloride synthesized from halite‐brine mush cold pressed at 150 MPa and 4.5 GPa, and natural rock salt mylonite from a salt glacier at Qom Kuh, central Iran). In addition, results from BIB‐cryo‐SEM on a gas shale and Boom Clay are also presented to show that the instrument is suitable for a large range of sedimentary rocks. For the first time, pore and grain fabrics of preserved host and reservoir rocks can be investigated at nm‐scale range over a representative elementary area. In comparison with the complementary and overlapping performances of the BIB‐SEM method with focused ion beam‐SEM and X‐ray tomography methods, the BIB cross‐sectioning enables detailed insights about morphologies of pores at greater resolution than X‐ray tomography and allows the production of large representative surfaces suitable for FIB‐SEM investigations of a specific representative site within the BIB cross‐section.     

NanoSIMS imaging of Bacillus spores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top‐sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift‐out. The lift‐out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top‐cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ～300 nm for top‐cut sections versus ～100 nm for electron‐transparent sections.     

Multiscale iterative voting for differential analysis of stress response for 2D and 3D cell culture models

Focused ion beam-scanning electron microscope (FIB-SEM) tomography is a powerful application in obtaining three-dimensional (3D) information. The FIB creates a cross section and subsequently removes thin slices. The SEM takes images using secondary or backscattered electrons, or maps every slice using X-rays and/or electron backscatter diffraction patterns. The objective of this study is to assess the possibilities of combining FIB-SEM tomography with cathodoluminescence (CL) imaging. The intensity of CL emission is related to variations in defect or impurity concentrations. A potential problem with FIB-SEM CL tomography is that ion milling may change the defect state of the material and the CL emission. In addition the conventional tilted sample geometry used in FIB-SEM tomography is not compatible with conventional CL detectors. Here we examine the influence of the FIB on CL emission in natural diamond and the feasibility of FIB-SEM CL tomography. A systematic investigation establishes that the ion beam influences CL emission of diamond, with a dependency on both the ion beam and electron beam acceleration voltage. CL emission in natural diamond is enhanced particularly at low ion beam and electron beam voltages. This enhancement of the CL emission can be partly explained by an increase in surface defects induced by ion milling. CL emission enhancement could be used to improve the CL image quality. To conduct FIB-SEM CL tomography, a recently developed novel specimen geometry is adopted to enable sequential ion milling and CL imaging on an untilted sample. We show that CL imaging can be manually combined with FIB-SEM tomography with a modified protocol for 3D microstructure reconstruction. In principle, automated FIB-SEM CL tomography should be feasible, provided that dedicated CL detectors are developed that allow subsequent milling and CL imaging without manual intervention, as the current CL detector needs to be manually retracted before a slice can be milled. Due to the required high electron beam acceleration voltage for CL emission, the resolution for FIB-SEM CL tomography is currently limited to several hundreds of nm in XY and up to 650 nm in Z for diamonds. Opaque materials are likely to have an improved Z resolution, as CL emission generated deeper in the material is not able to escape from it.     

Towards the imaging of Weibel–Palade body biogenesis by serial block face‐scanning electron microscopy

Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face‐scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three‐dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre‐scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three‐dimensional analysis. Here, we explore focused ion beam facilitated serial block face‐scanning electron microscopy to study the endothelial cell–specific storage organelles, the Weibel–Palade bodies, during their biogenesis at the Golgi apparatus. Weibel–Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face‐scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel–Palade bodies and Golgi stacks which became conceivable in large‐scale three‐dimensional data. We demonstrate that serial block face‐scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.     

Focused ion beam manipulation and ultramicroscopy of unprepared cells

The focused ion beam (FIB) technique of nanomachining combined with simultaneous scanning electron microscopy (SEM) was used for submicron manipulation and imaging of unprepared (fresh) cells to demonstrate the potentiality of the FIB/SEM technique for ultramicroscopic studies. Sectioning at the nanoscale level was successfully performed by means of ion beam-driven milling operations that reveal the ultrastructure of fresh yeast cells. The FIB/SEM has many advantages over other ultramicroscopy techniques already applied for unprepared/fresh biological samples.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Site-specific structural investigations of oxidized nickel samples modified by plasma erosion processes

A focused ion beam was employed for local target preparation for EBSD analysis. The volume of the ion‐solid interaction is well below 50 nm at glancing incidence for metallic and transition metal oxide samples. Therefore, focused ion beam can successfully be used for electron backscatter diffraction (EBSD) sample preparation. The sample investigated consists of Ni covered with a NiO layer of ～5 μm thickness. Focused ion beam cross‐sectioning of these layers and subsequent electron imaging in addition to EBSD maps shows a bimodal structure of the oxide layer. In order to test the potential of such oxidized samples as electrode materials, single spark erosion experiments were performed. The erosion craters have diameters up to 40 μm and have a depth corresponding to the thickness of the oxide layer. In addition, a deformation zone produced by thermoshock accompanies the formation of the crater. This deformation zone was further investigated by EBSD analysis using a new way of sample preparation employing the focused ion beam technology. This target preparation routine is called Volume of Interest Transfer and has the potential of providing a full three‐dimensional characterization.     

Evolution of residual stress and crack morphologies during 3D FIB tomographic analysis of alumina

Three‐dimensional focused ion beam (FIB) tomography is increasingly being used for 3D characterization of microstructures in the 50 nm–20 μm range. FIB tomography is a destructive, invasive process, and microstructural changes may potentially occur during the analysis process. Here residual stress and crack morphologies in single‐crystal sapphire samples have been concurrently analyzed using Cr³⁺ fluorescence spectroscopy and FIB tomography. Specifically, maps of surface residual stress have been obtained from optically polished single‐crystal alumina [surface orientation (1 ī 0 2)], from FIB milled surface trenches, from Vickers micro‐indentation sites (loads 50 g–300 g), and from Vickers micro‐indentation sites during FIB serial sectioning. The residual stress maps clearly show that FIB sputtering generates residual stress changes. For the case of the Vickers micro‐indentations, FIB sputtering causes significant changes in residual stress during the FIB tomographic serial sectioning. 3D reconstruction of the crack distribution around micro‐indentation sites shows that the cracks observed are influenced by the location of the FIB milled surface trenches due to localized stress changes.     

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.     

Focussed ion beam milling at grazing incidence angles

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.     

Image quality evaluation for FIB-SEM images

Focused ion beam scanning electron microscopy (FIB-SEM) tomography is a serial sectioning technique where an FIB mills off slices from the material sample that is being analysed. After every slicing, an SEM image is taken showing the newly exposed layer of the sample. By combining all slices in a stack, a 3D image of the material is generated. However, specific artefacts caused by the imaging technique distort the images, hampering the morphological analysis of the structure. Typical quality problems in microscopy imaging are noise and lack of contrast or focus. Moreover, specific artefacts are caused by the FIB milling, namely, curtaining and charging artefacts. We propose quality indices for the evaluation of the quality of FIB-SEM data sets. The indices are validated on real and experimental data of different structures and materials.     

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three‐dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze‐fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block‐face, SBF‐SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.     

Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.     

FIB/SEM characterization of carbon-based fibers

The aim of this paper is to show how a focused ion beam combined with a scanning electron microscope (FIB/SEM machine) can be adopted to characterize composite fibers with different electrical behavior and to gain information about their production and modification. This comparative morphology investigation is carried out on polyacrylonitrile (PAN) carbon fibers and their chemical precursor (the oxidized PAN or oxypan) which has different electrical properties. Fibers are imaged by electron and ion beams and sectioned by the focused ion beam (FIB). A sample of oxypan fibers processed by a radio frequency (RF) plasma is also investigated and the role of the conductive carbon layer around their unmodified, insulating bulk is discussed. A suitable developed edge detection technique (EDT) on electron, ion images, and after the FIB sectioning, provides quantitative information about the thickness of the created layer.     

A technique for improved focused ion beam milling of cryo-prepared life science specimens

The combination of focused ion beam and scanning electron microscopy with a cryo‐preparation/transfer system allows specimens to be milled at low temperatures. However, for biological specimens in particular, the quality of results is strongly dependent on correct preparation of the specimen surface. We demonstrate a method for deposition of a protective, planarizing surface layer onto a cryo‐sample, enabling high‐quality cross‐sectioning using the ion beam and investigation of structures at the nanoscale.     

Study of hyperpigmentation in human skin disorder using different electron microscopy techniques

There is a global trend of increase in the demand for three‐dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block‐face scanning electron microscopy and focused ion beam—scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis‐endocytosis theory of pigment in human skin.     

Surface damage induced by FIB milling and imaging of biological samples is controllable

Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.