Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.     

Focused ion beam/scanning electron microscopy studies of Porcellio scaber (Isopoda, Crustacea) digestive gland epithelium cells

The focused ion beam (FIB) was used to prepare cross sections of precisely selected regions of the digestive gland epithelium of a terrestrial isopod P. scaber (Isopoda, Crustacea) for scanning electron microscopy (SEM). The FIB/SEM system allows ad libitum selection of a region for gross morphologic to ultrastructural investigation, as the repetition of FIB/SEM operations is unrestricted. The milling parameters used in our work proved to be satisfactory to produce serial two-dimensional (2-D) cuts and/or three-dimensional (3-D) shapes on a submicrometer scale. A final, cleaning mill at lower ion currents was employed to minimize the milling artifacts. After cleaning, the milled surface was free of filament- and ridge-like milling artifacts. No other effects of the cleaning mill were observed.     

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.     

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.     

Ultrastructure of the Lyonet's glands in larvae of Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae).

The Lyonet's gland is found in Lepidoptera larvae, close to the excretory duct of the silk gland. The role played by this gland is still uncertain. This work aims to describe the ultrastructure of the Lyonet's gland in Diatraea saccharalis larvae, offering suggestions regarding its possible function. The insects were reared under laboratory-controlled conditions. The glands were conventionally prepared for transmission (TEM) and scanning (SEM) electron microscopy. SEM showed that Lyonet's glands are paired small structures located in the ventral side of the head. They are composed by clustered long cells resembling leaves. Under TEM observations, each cell is surrounded by a thin basal lamina and contains large stellate nucleus. The cytoplasm presents large and empty canaliculi with small microvilli. The basal plasma membrane forms numerous infoldings where numerous and well-developed mitochondria are concentrated. The cytoplasmic membrane system is poorly developed. Our ultrastructural results suggest that the Lyonet's gland in D. saccharalis larvae may be involved in the uptake of small molecules from the hemolymph; no morphological evidences of macromolecules synthesis and secretion were noticed. The detection of nerve fibers in the gland suggest a neural control for the glandular cell function.     

Structural and ultrastructural features of the Malpighian tubules of Dolycoris baccarum (Linnaeus 1758), (Heteroptera: Pentatomidae)

The morphology and ultrastructure of the Malpighian tubules of Dolycoris baccarum were analyzed by scanning (SEM) and transmission (TEM) electron microscopy in order to determine their functional organization. The Malpighian tubules are compared with similar structures of other insects based on cell structure and functional organization. The Malpighian tubules of D. baccarum extend from the midgut–hindgut region of the digestive tract. The Malpighian tubules are divided into two regions: the proximal segment is short and flattened and the distal segment is long, stringy in shape and free in hemolymph. The tubules are generally long and narrow. There is a large number of trachea around the tubules. They consist of a single layer of epithelial cells. It is observed in the TEM observation that the epithelial cells have numerous microvilli at the apical side of the cells. At the basal side of the cells, there is a great number of membrane foldings and mitochondria among them. Besides some spherites, mitochondria, lysosome‐like bodies, and large or small granules can be distinguished in the cells. With this study, we aimed to demonstrate the ultrastructure of the Malpighian tubules of D. baccarum and differences or similarities with other species.     

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.     

聚焦离子束电子束系统及其在生物学和医学上的应用

聚焦离子束电子束系统在材料失效分析、纳米材料结构表征与性能分析以及纳米器件研制等方面发挥着重要作用。近年来该系统在生物学和医学领域的应用日益受到人们的重视。本文介绍聚焦离子束电子束系统的组成、性能、相关功能及其在生物学和医学上的若干应用,包括透射电镜生物样品制备、细胞和组织内部结构观察与三维重构等。     

Seasonal ultrastructural changes in apocrine gland cells in back skin of male brown bears (Ursus arctos)

Oily secretions from the back skin are involved in the marking behavior of male brown bears (Ursus arctos), and apocrine glands in back skin are activated during the breeding season. Here, we investigated seasonal changes in the intracellular organelles of apocrine gland cells in the back skin of male brown bears using transmission electron microscopy (TEM) and osmium‐maceration scanning electron microscopy (OM‐SEM). The morphological features of mitochondria and intracellular granules, and secretory mechanisms obviously differed between breeding and non‐breeding seasons. The TEM findings showed that contents of low‐density granules were released into the glandular lumen by frequent exocytosis, and sausage‐shaped mitochondria were located in the perinuclear region during the non‐breeding season. In contrast, high‐density granules appeared in the apical region and in projections during the breeding season, and swollen mitochondria and lysosome‐like organelles separating into high‐density granules were located in the perinuclear region. The OM‐SEM findings revealed swollen mitochondria with only a few partially developed cristae, and small mitochondria with cristae shaped like those in swollen mitochondria in the apical regions during the breeding season. These findings indicated that the small mitochondria corresponded to the high‐density granules identified by TEM. These findings suggested that mitochondria in apocrine gland cells swell, degenerate, fracture into small pieces, and are finally released by apocrine secretions during the breeding season. Small mitochondria released in this secretory manner might function as the source of chemical signals in the oily secretions of brown bears during the breeding season.     

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

Improved preservation of phospholipid-rich multilamellar bodies in conventionally embedded mammalian lung tissue—an electron spectroscopic study

Different conventional methods of tissue processing were studied to determine the extent to which phospholipid-rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half-saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde-paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm. Differences in the phosphorus distribution among several types of lipid bodies of alveolar epithelial type II cells were studied by means of electron spectroscopic imaging and electron energy-loss spectroscopy. Multilamellar bodies gave phosphorus signals which were significantly higher than those obtained from granular regions of composite bodies, whereas homogeneous bodies gave phosphorus signals which were even lower than those obtained from mitochondria, endoplasmic reticulum membranes or ribosomes.     

Cytochemical investigation of the digestive gland of two strombidae species (Strombus gigas and Strombus pugilis) in relation to the nutrition

Strombus gigas and Strombus pugilis are threatened species and aquaculture represents a good alternative solution to the fishing. In this study, we highlighted the intracellular digestion process in the digestive gland of two Strombidae species, S. gigas and Strombuspugilis, by the cytochemical characterization of two lysosomal enzymes: acid phosphatase and arylsulfatase. In order to check the efficiency of artificial food digestion, we conducted the characterization on freshly collected, starved and artificially fed individuals of S. pugilis. TEM observations of digestive gland sections from freshly collected individuals of both species revealed the presence of acid phosphatase and arylsulfatase activity mostly located in the apical third of digestive cells. Both enzymes were also detected in artificially fed individuals. In response to the starvation, acid phosphatase is not produced anymore by digestive cells, while arylsulfatase is still present. To our knowledge, this is the first cytochemical validation of intracellular digestion of artificial food in Strombidae. This study highlights the intracellular digestion of artificial food developed for Strombidae aquaculture. Moreover, we have shown that the lysosomal activity could be used as a feed index. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.     

Histological and ultrastructural features of the rectum in Poecilimon cervus Karabağ, 1950 (Orthoptera: Tettigoniidae)

The morphology and ultrastructure of the rectum in Poecilimon cervus Karaba?, 1950 (Orthoptera, Tettigoniidae) were analyzed by light microscope, scanning (SEM) and transmission electron microscopes (TEM). The rectum is the final part of the digestive tract that plays an important role in water reabsorption in insects and so provides osmoregulation. In the transverse sections, six rectal pads and columnar epithelium can be distinguished. The cuticular intima lines the lumen at the apical side of the epithelium. In the cytoplasm, there are numerous mitochondria, some endocytic vesicles, secreting vesicles whose sizes differ according to the area in the cell, and a nucleus with globular in shape. With this study, we aimed to demonstrate the ultrastructure of the rectum of P. cervus and differences or similarities of with other species.     

The photoreceptive cells of the pineal gland in adult zebrafish (Danio rerio)

The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions.     

Morphology and histology of P. argentinus (Crustacea, Decapoda, Caridea) digestive tract.

This work describes the morphology and histology of the P. argentinus digestive tract. The foregut comprises the mouth, oesophagus, and stomach and is lined by a simple cylindrical epithelium overlain by cuticle. There are tegumental glands in the oral region and in the first portion of the oesophagus and of the hindgut. The cardiac stomach is an oval dorsal sac in the cephalothorax and has no calcified structures. The pyloric stomach comprises an upper chamber and a lower gland filter. The filter consists of an outer row of elongated setae and an inner row of dorsally curved setae forming longitudinal channels 16-18 microm wide. The midgut runs from the dorsal chamber of the pyloric stomach to the sixth abdominal somite without caeca. The hindgut runs from the sixth abdominal somite to the ventral anus. The mid-gut epithelium comprises dominant cylindrical cells and small undifferentiated cells in the first portion. The hindgut wall presents longitudinal folds, conspicuous muscular bundles, and a folded cuticle. The digestive tract of P. argentinus is basically similar to that of most of decapods. The absence of calcified structures in the stomach and the width of the longitudinal channels in the filter are related to the predominantly detritivorous diet.     

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 preparation and SEM investigations for three-dimensional analysis of cell cultures on microneedle arrays

We report the investigation of the interfaces between microneedle arrays and cell cultures in patch-on-chip systems by using Focused Ion Beam (FIB) preparation and Scanning Electron Microscopy (SEM). First, FIB preparations of micro chips are made to determine the size and shape of the designed microneedles. In this essay, we investigate the cell-substrate interaction, especially the cell adhesion, and the microneedle's potential cell penetration. For this purpose, cross-sectional preparation of these hard/soft hybrid structures is performed by the FIB technology. By applying the FIB technology followed by high-resolution imaging with SEM, new insights into the cell-substrate interface can be received. One can clearly distinguish between cells that are only in contact with microneedles and cells that are penetrated by microneedles. A stack of slice images is collected by the application of the slice-and-view setup during FIB preparation and is used for three-dimensional reconstruction of cells and micro-needles.     

Minimization of focused ion beam damage in nanostructured polymer thin films

Focused ion beam (FIB) instruments have proven to be an invaluable tool for transmission electron microscopy (TEM) sample preparation. FIBs enable relatively easy and site-specific cross-sectioning of different classes of materials. However, damage mechanisms due to ion bombardment and possible beam heating effects in materials limit the usefulness of FIBs. Materials with adequate heat conductivity do not suffer from beam heating during FIB preparation, and artifacts in materials such as metals and ceramics are primarily limited to defect generation and Ga implantation. However, in materials such as polymers or biological structures, where heat conductivity is low, beam heating can also be a problem. In order to examine FIB damage in polymers we have undertaken a systematic study by exposing sections of a PS-b-PMMA block copolymer to the ion beam at varying beam currents and sample temperatures. The sections were then examined by TEM and scanning electron microscopy (SEM) and analyzed using electron energy loss spectroscopy (EELS). Our empirical results show beam heating in polymers due to FIB preparation can be limited by maintaining a low beam current (≤100 pA) during milling.     

Fine structure of the vomeronasal and septal olfactory epithelia and of glandular structures.

The vomeronasal and septal olfactory organs are two neurosensory structures in the mammalian nasal septum which are poorly understood relative to the main olfactory system. The vomeronasal organ is a paired, blind-ending tubular structure that opens rostrally into the nasal cavity in some species and into the incisive ducts in others. When present in mammals, the septal olfactory organ is an island of olfactory mucosa positioned such that it is in the primary air pathway in the caudal portion of the nasal cavity. Mammalian nasal glands, with a diverse histochemical and ultrastructural morphology, secrete a variety of substances onto the mucosal surface. One of these substances, odorant binding protein, localized in bovine nasal glands and lateral nasal glands of rodents, may be important in the capture and conveyance of odorant molecules to olfactory receptors. The objectives of this paper are to present original data while reviewing the literature on the ultrastructure of vomeronasal and septal olfactory neuroepithelia, and of vomeronasal, bovine nasal, and lateral nasal glands. Nasal tissues from pigs, calves, and hamsters were prepared for electron microscopy. Neurosensory epithelia of the porcine vomeronasal organ and the hamster septal olfactory organ are similar to that described for the vomeronasal and septal olfactory organs of other mammals. Bovine nasal and rodent lateral nasal glands consist of subregions which differ morphologically; the most abundant acinar cell type in the bovine nasal gland contains lightly electron dense secretory granules while that of the rodent lateral nasal gland contains both small electron dense and large, electron lucent granules. The porcine vomeronasal gland contains numerous small, dense granules of a diverse morphology.