Early interpretations of biological structures using film-based transmission electron microscopy (TEM) images were based on the analysis of two-dimensional TEM images, or by the painful reconstruction and analysis of serial TEM image stacks. In today’s era of computer-centric, three-dimensional imaging, it is hard to imagine a time when scientists and microscopists were forced to reconstruct and interpret actual stacks of photographs generated from serially sectioned tissue. While TEM images provided a quantum leap forward in our understanding of the structures inside cells, serial sectioning is a very specialized technique that presents several unique challenges. It requires a number of steps to be completed flawlessly to obtain results with high scientific value, and each step demands considerable operator skill. The technique is made more challenging by the unavailability of feedback at intermediate steps, requiring completion of the entire process before the quality of the result can be evaluated. Problems with fixation, staining, knife marks, etc., remain undiscovered until the finished sample is examined with the microscope. If problems exist, the entire preparation process must be repeated, presuming the sample was not unique and another with similar characteristics is readily available.
In DualBeam™ (FEI, Hillsboro, OR) instruments, the electron beam and the ion beam are specifically configured to facilitate high-resolution scanning electron microscopy (SEM) imaging of the surface milled by the FIB. Both beams are focused at the same point (the coincident point) on the sample with the electron beam vertical and the ion beam off vertical by approximately 50 degrees. Typically, the sample is tilted so that its surface is perpendicular to the ion beam and the milled surface is thus also perpendicular to the sample surface. In this configuration, the milled surface is presented to the electron beam for immediate imaging without repositioning the sample. In fact, the SEM can be used to view the surface as milling proceeds, providing immediate visual feedback on the progress of the milling operation, which can be critical in situations where it is important to stop milling when the feature of interest is reached.
DualBeam milling process
Scientists are actively refining the process for using DualBeam instruments as a new tool for the generation of high-resolution three-dimensional images of biological tissues and cell structures. In most cases, the sample is initially fixed, stained, and embedded following protocols similar to those used for standard TEM sectioning. This typically consists of fixation in a buffered aldehyde solution, postfixation staining with osmium tetroxide (osmication), and then embedding in conventional resin. Osmium tetroxide reacts primarily with lipids and adds contrast in biological tissues by combining with membrane structures and increasing their electron scattering ability. The introduction of this heavy metal stain into biological samples has different, but predictable, effects in both TEM and SEM imaging.
Embedded samples are then mounted to a standard SEM stub with silver paint and coated with a conductive metal, like platinum-palladium, to reduce charge buildup. Alternately, cryo electron microscopy preparation techniques have produced good fixation and imaging results.1
Figure 1 - Diagram of DualBeam sample configuration. The white beam and arrows indicate the FIB milling region, and the gray beam indicates the orientation of the SEM imaging beam.2 (Reproduced with permission from Ref. 2.)
Once mounted and loaded in the DualBeam system, controlled amounts of material are removed by the focused ion beam. After each material removal sequence, an SEM image is acquired (Figure 1). Repeating this process through a given volume of tissue generates a series of two-dimensional images with nanometer scale (>5 nm) resolution.
The FIB portion of the DualBeam can be programmed to remove material from the sample in sections as thin as 5 nm. The thinnest serial sections possible with a diamond knife and ultramicrotome are about 30 nm; thus Dual-Beam milling offers up to a 6× improvement in resolution in the sectioning (Z) direction. The thickness of the section also affects lateral resolution (X,Y). In a TEM, overlap of structural elements in the beam direction introduces ambiguity in the resulting two-dimensional images. Beam penetration has similar effects in SEM imaging. As it penetrates below the sample surface it also spreads laterally. The further the beam penetrates, the less specific (both laterally and vertically) is the information in the signals it generates to the point of incidence on the sample surface. Accurately controlling the depth of penetration of the electron beam into the sample is important to final image quality, since it determines the resolution of the images in all three dimensions. Ideally, the electron beam should penetrate to a depth less than that of the thickness of material removed by each milling cycle.
The primary mechanism for controlling penetration depth is the regulation of the accelerating voltage. Lower accelerating voltages result in less sample penetration by the electron beam. Some calibration may be required to accurately determine beam penetration depths on different materials. The need for precise control of penetration depth becomes more important as the interimage distance is reduced.
Figure 2 - Three-dimensional reconstruction of biological tissue structure using Slice and View software. Black and white images indicate a stack of serial images acquired with the SEM after FIB milling. Color image (bottom right) shows software generated in the three-dimensional model.3 (Reproduced with permission from Ref. 3.)
After material is removed, an image is collected using either the secondary electron or backscattered electron signal. The acquired series of images is then registered and reconstructed using software, such as FEI Slice and View, to generate highly accurate three-dimensional models of the imaged structures (Figure 2).
In addition to improving intrasection resolution, this technique provides greater flexibility in the choice of region for investigation and allows high resolutions over larger fields of view. In situ FIB milling also eliminates the need to manually cut sections on an ultramicrotome, arrange them on a grid, and move them to the microscope. Elimination of ultramicrotomy means there are no mechanical distortions or artifacts induced by mechanical sectioning. Additionally, the entire milling and image acquisition process can be automated, to save time and reduce chances for operator-induced errors. Automated milling/image acquisition cycle times are on the order of 3 min for a 10 μm × 10 μm area.
Applications and future direction
Figure 3 - a) Image of a plunge frozen yeast cell sectioned and imaged using the DualBeam.3 b) Image of neural tissue; the inset with arrowheads indicates a symmetric and asymmetric synapse.2 (Reproduced with permission from Ref. 2.)
The method has been used effectively to study structures such as neuronal structure, and connectivity, cross-sections of yeast cells, cell junctions, and other subcellular structures, producing images of very high quality and resolution (Figure 3). The results it provides are prompting scientists to rethink original interpretations of some of the classic subcellular components, such as mitochondrial membrane structures and the relationships between organelles and the endoplasmic reticulum.
DualBeam serial sectioning is also well suited for analyzing the interaction between cells and hard materials, such as metallic probes, implants, and nanodevices. These interactions are often challenging, or impossible, to investigate with mechanical sectioning. Because the material is milled, rather than sliced, the technique does not experience issues like fracturing or distortion of the section that result from the mechanical forces exerted during cutting.
The increased use of the DualBeam SEM/FIB system for investigation of biological tissue offers several significant advantages to today’s scientists and researchers. Electron microscopy provides at least an order of magnitude improvement in resolution over optical microscopy, and SEM/FIB milling allows for thinner sections for even better resolution. The combination of automation with advanced reconstruction software greatly simplifies the generation of three-dimensional images. As this technique gains recognition and acceptance in the scientific community, it will likely be combined with other methods to increase our understanding of biological structure and function. An increasing number of applications are benefiting from the rapid, high-quality three-dimensional results produced by this method.
- Lich, B. Site specific three-dimensional structural analysis in tissues and cells using automated DualBeam Slice and View. Microscopy Today Mar 2007, 26–30.
- Knott, G.; Marchman, H.; Wall, D.; Lich, B. Serial sectioning scanning electron microscopy of adult brain tissue using focused ion beam milling. J. Neurosci. Mar 19, 2008, 2959–64.
- Heymann, J.A.; Hayles, M.; Gestmann, I.; Giannuzzi, L.A.; Lich, B.; Subramaniam, S. Site specific 3D imaging of cells and tissues with a dual beam microscope. J. Struct. Biol. Jul 2006, 155(1), 63–73.
Dr. Lich is Marketing Manager for SEM/SDB, Life Sciences Div., FEI, Netherlands, Achtseweg Noord 5, Building AAE, 5651 GG Eindhoven/Acht, The Netherlands; tel.: +31 651 572 164; e-mail: Ben.firstname.lastname@example.org.