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.

Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼500nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.

New imaging techniques applied to Paleogene radiolaria

A simple and time efficient technique to illustrate specimens is described and demonstrated with Paleogene radiolarians. This method produces Scanning Electron Microscope (SEM) and composite focal depth Transmitted Light Microscope (TLM) images for single radiolarian specimens. We propose the use of this technique to clarify radiolarian taxonomy. This technique has distinct advantages over previously published time consuming techniques that can also require toxic materials.

FIB-SEM tomography in biology.

Three-dimensional information is much easier to understand than a set of two-dimensional images. Therefore a layman is thrilled by the pseudo-3D image taken in a scanning electron microscope (SEM) while, when seeing a transmission electron micrograph, his imagination is challenged. First approaches to gain insight in the third dimension were to make serial microtome sections of a region of interest (ROI) and then building a model of the object. Serial microtome sectioning is a tedious and skill-demanding work and therefore seldom done. In the last two decades with the increase of computer power, sophisticated display options, and the development of new instruments, an SEM with a built-in microtome as well as a focused ion beam scanning electron microscope (FIB-SEM), serial sectioning, and 3D analysis has become far easier and faster.Due to the relief like topology of the microtome trimmed block face of resin-embedded tissue, the ROI can be searched in the secondary electron mode, and at the selected spot, the ROI is prepared with the ion beam for 3D analysis. For FIB-SEM tomography, a thin slice is removed with the ion beam and the newly exposed face is imaged with the electron beam, usually by recording the backscattered electrons. The process, also called "slice and view," is repeated until the desired volume is imaged.As FIB-SEM allows 3D imaging of biological fine structure at high resolution of only small volumes, it is crucial to perform slice and view at carefully selected spots. Finding the region of interest is therefore a prerequisite for meaningful imaging. Thin layer plastification of biofilms offers direct access to the original sample surface and allows the selection of an ROI for site-specific FIB-SEM tomography just by its pronounced topographic features.

Examination of Heterogeneous Crossing Sequences Between Toner and Rollerball Pen Strokes by Digital Microscopy and 3-D Laser Profilometry

The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner. With the mass marketing of laser printers and the popularity of rollerball pens, the determination of line crossing sequences between such instruments is encountered by forensic document examiners. This type of crossing presents difficulties with optical microscopic line crossing techniques involving ballpoint pens or gel pens and toner (1-4). Indeed, the rollerball's aqueous ink penetrates through the toner and is absorbed by the fibers of the paper, leaving the examiner with the impression that the toner is above the ink even when it is not (5). Novotny and Westwood (3) investigated the possibility of determining aqueous ink and toner crossing sequences by microscopic observation of the intersection before and after toner removal. A major disadvantage of their study resides in destruction of the sample by scraping off the toner line to see what was underneath. The aim of this research was to investigate the ways to overcome these difficulties through digital microscopy and three-dimensional (3-D) laser profilometry. The former was used as a technique for the determination of sequences between gel pen and toner printing strokes, but provided less conclusive results than that of an optical stereomicroscope (4). 3-D laser profilometry, which allows one to observe and measure the topography of a surface, has been the subject of a number of recent studies in this area. Berx and De Kinder (6) and Schirripa Spagnolo (7,8) have tested the application of laser profilometry to determine the sequence of intersections of several lines. The results obtained in these studies overcome disadvantages of other methods applied in this area, such as scanning electron microscope or the atomic force microscope. The main advantages of 3-D laser profilometry include the ease of implementation of the technique and its nondestructive nature, which does not require sample preparation (8-10). Moreover, the technique is reproducible and presents a high degree of freedom in the vertical axes (up to 1000 μm). However, when the paper surface presents a given roughness, if the pen impressions alter the paper with a depth similar to the roughness of medium, the results are not always conclusive (8). It becomes difficult in this case to distinguish which characteristics can be imputed to the pen impressions or the quality of the paper surface. This important limitation is assessed by testing different types of paper of variable quality (of different grammage and finishing) and the writing pressure. The authors will therefore assess the limits of 3-D laser profilometry technique and determine whether the method can overcome such constraints. Second, the authors will investigate the use of digital microscopy because it presents a number of advantages: it is efficient, user-friendly, and provides an objective evaluation and interpretation.

The biogenic origin of needle fibre calcite

Needle fibre calcite is one of the most ubiquitous habits of calcite in vadose environments (caves deposits, soil pores, etc.). Its origin, either through inorganic, indirect or direct biological processes, has long been debated. In this study, investigations at 11 sites in Europe, Africa and Central America support arguments for its biogenic origin. The wide range of needle morphologies is the result of a gradual evolution of the simplest type, a rod. This rod is the elementary brick which, by aggregation and welding, builds more complex needles. The absence of cross-welded needles implies that they are welded in a mould, or under a longitudinal and unidirectional constraint, before being released inside the soil pores. The difference between the lengthening of the needles and the c axis can be explained by the existence of needles observed under a scanning electron microscope in organic sleeves, which can act as a mould during rod growth. Complex morphologies with epitaxial outgrowths on straight rods cannot have grown entirely inside organic microtubes; they must result from soil diagenesis after the release of straight rods in a soil-free medium. Whisker crystals are interpreted as the result of growth and coalescence of euhedral crystals on a rod. Rhomb chains are considered to be the consequence of successive epitaxial growth steps on a needle during variations in growth conditions. Isotopic signatures for needle fibre calcite vary from -16.63[per mille] to +1.10[per mille] and from -8.63[per mille] to -2.25[per mille] for Delta13C and Delta18O, respectively. The absence of high Delta18O values for needle fibre calcite precludes a purely physicochemical origin (evaporative) for this particular habit of calcite. As epitaxial growth cannot precipitate in the same conditions as initial needles, needle fibre calcite stable isotopic signatures should be used with caution as a proxy for palaeoenvironmental reconstructions. In addition, it is suggested that the term needle fibre calcite should be kept for the original biogenic form. The other habit should be referred to as epitaxial forms of needle fibre calcite.

Surface reconstruction from microscopic images in optical lithography.

This paper presents a method to reconstruct 3D surfaces of silicon wafers from 2D images of printed circuits taken with a scanning electron microscope. Our reconstruction method combines the physical model of the optical acquisition system with prior knowledge about the shapes of the patterns in the circuit; the result is a shape-from-shading technique with a shape prior. The reconstruction of the surface is formulated as an optimization problem with an objective functional that combines a data-fidelity term on the microscopic image with two prior terms on the surface. The data term models the acquisition system through the irradiance equation characteristic of the microscope; the first prior is a smoothness penalty on the reconstructed surface, and the second prior constrains the shape of the surface to agree with the expected shape of the pattern in the circuit. In order to account for the variability of the manufacturing process, this second prior includes a deformation field that allows a nonlinear elastic deformation between the expected pattern and the reconstructed surface. As a result, the minimization problem has two unknowns, and the reconstruction method provides two outputs: 1) a reconstructed surface and 2) a deformation field. The reconstructed surface is derived from the shading observed in the image and the prior knowledge about the pattern in the circuit, while the deformation field produces a mapping between the expected shape and the reconstructed surface that provides a measure of deviation between the circuit design models and the real manufacturing process.

Calcitic nanofibers in soils and caves : a putative fungal contribution to carbonatogenesis

The origin of soil mineralized nanofibres remains controversial. It is attributed to either biogenic factors or physicochemical processes. Scanning electron microscope and transmission electron microscope observations show that nanofibres could originate from the breakdown of fungal hyphae, especially its cell wall. It is hypothesized that during the decay of organic matter, cell wall microfibrils are released in the soil where they are exposed to mineralizing pore fluids, leading to their calcitic pseudomorphosis and/or are used as a template for calcite precipitation. When associated with needle fibre calcite bundles, nanofibres could indicate the relict of an organic sheath in which calcite has precipitated. This paper emphasizes the important roles of both organic matter and fungi in carbonatogenesis, and consequently in the soil carbon cycle.

A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

Correlative light and electron microscopy in parasite research.

The interaction of a parasite and a host cell is a complex process, which involves several steps: (1) attachment to the plasma membrane, (2) entry inside the host cell, and (3) hijacking of the metabolism of the host. In biochemical experiments, only an event averaged over the whole cell population can be analyzed. The power of microscopy, however, is to investigate individual events in individual cells. Therefore, parasitologists frequently perform experiments with fluorescence microscopy using different dyes to label structures of the parasite or the host cell. Though the resolution of light microscopy has greatly improved, it is not sufficient to reveal interactions at the ultrastructural level. Furthermore, only specifically labeled structures can be seen and related to each other. Here, we want to demonstrate the additional value of electron microscopy in this area of research. Investigation of the different steps of parasite-host cell interaction by electron microscopy, however, is often hampered by the fact that there are only a few cells infected, and therefore it is difficult to find enough cells to study. A solution is to profit from low magnification, hence large overview, and specific location of the players by fluorescence labels in a light microscope with the high power resolution and structural information provided by an electron microscope, in short by correlative light and electron microscopy.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells.

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser.

BACKGROUND and OBJECTIVE: A non-touch laser-induced microdrilling procedure is studied on mouse zona pellucida (ZP). STUDY DESIGN/MATERIALS and METHODS: A 1.48-microns diode laser beam is focused in a 8-microns spot through a 45x objective of an inverted microscope. Mouse zygotes, suspended in a culture medium, are microdrilled by exposing their ZP to a short laser irradiation and allowed to develop in vitro. RESULTS: Various sharp-edged holes can be generated in the ZP with a single laser irradiation. Sizes can be varied by changing irradiation time (3-100 ms) or laser power (22-55 mW). Drilled zygotes present no signs of thermal damage under light and scanning electron microscopy and develop as expected in vitro, except for a distinct eight-shaped hatching behavior. CONCLUSION: The microdrilling procedure can generate standardized holes in mouse ZP, without any visible side effects. The hole formation can be explained by a local photothermolysis of the protein matrix.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.