Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.     

Focussed ion beam milling and scanning electron microscopy of brain tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.     

Observations of sporulation in Fonsecaea and Phialophora by scanning electron microscopy

An extracellular lipase produced by Thermomyces lanuginosus in palm fruit chaff infusion broth at 45 °C after 6 days of incubation was purified by a combination of ultrafiltration, ethanol precipitation and fractionation on DEAE-cellulose and gel-filtration on Sephadex G.200. A single peak of lipase activity with a tenfold increase in the activity of the enzyme was obtained. The partially purified T. lanuginosus lipase had a recovery value of 25%. Attempts to purify this enzyme further led to an almost complete loss of activity. The lipase had a pH optimum of 6.5 and peak activity at 40 °C. It readily hydrolysed both natural and synthetic triglycerides at 40 °C with optimal activities recorded on palm oil and triolein respectively.     

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.     

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy

HeLa metaphase chromosomes were exammed by means of in lens field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.     

Observations of Microsporum canis with cryoscanning and scanning electron microscopy

The observations of Microsporum canis with cryoscanning and scanning electron microscopy without fixation and dehydration were reported. In the former an almost native state was observed through showing some fuzzy outlines due to frost; in the latter it was shown that marked shrinkage and distortion had occured. There were many granules on the surface of the macroconidia though their function is uncertain.     

中国海区几种隐藻类鞭毛藻的扫描电镜观察

报道了来自香港吐露港、中国长江口及厦门港的3个属的3种隐藻及1个变种, 即半片藻Hemiselmis sp. Novarino、伸长斜片藻Plagioselmis prolonga Butcher ex Novarino, Lucas ＆ Morrall、伸长斜片藻北方变种Plagioselmis prolonga var. nordica Novarino, Lucas ＆ Morrall、尖尾全沟藻Teleaulax acuta （Butcher） Hill, 并对每个种类的分类特征、生态分布进行描述, 同时提供每个种的光镜和扫描电镜照片。其中, 半片藻属Hemiselmis Parke是中国海区首次记录的属, 而伸长斜片藻Plagioselmis prolonga和尖尾全沟藻Teleaulax acuta可以引发赤潮。     

井上蛀果斑螟触角感器的扫描电镜观察

利用扫描电镜对井上蛀果斑螟Assarainouei Yamanaka雌雄成虫触角及触角感器的超微结构、类型和分布进行详尽观察。结果表明,井上蛀果斑螟触角上主要分布有9种类型的感器,其中毛形感器2种,锥形感器、刺形感器、耳形感器、腔锥形感器、腔乳头状感器、鳞形感器及Bohm氏鬃毛均1种。其中的腔乳头状感器只在雌虫触角上有分布,雄虫触角鳞形感器及末端毛形感器的数量均多于雌虫。     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

悬铃木方翅网蝽触角感器扫描电镜观察

利用扫描电镜对悬铃木方翅网蝽Corythucha ciliata(Say)雌、雄成虫触角背面和腹面进行观察。结果表明:悬铃木方翅网蝽触角为棒状,共4节,分为柄节、梗节和鞭节。触角上共有4种感器,分别为刺型感器、锥形感器、毛型感器和芽型感器;这些感器不存在性二型现象。其中,刺型感器分为大刺型感器和小刺型感器2种类型;芽型感器首次在异翅亚目昆虫触角上发现。雄成虫触角感器数量明显多于雌成虫,不同类型的感器在触角各节上的数量与分布各不相同。     

Observations of bacterial microcolonies on the surface of ferromanganese nodules from blake plateau by scanning electron microscopy

Examination of the surface of freshly collected ferromanganese nodules by scanning electron microscopy revealed the presence of microcolonies of rod- and coccus-shaped bacteria which appeared to be anchored to the nodule surface by slime. The attachment of microcolonies by slime to the surface of freshly collected nodules argues against their being contaminants introduced during nodule collection or processing. These results corroborate cultural and biochemical detection of bacteria on ferromanganese nodules.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Natural populations of bacteria in Lake Kinneret: Observations with scanning electron and epifluorescence microscopy

The bacterioplankton assemblage in Lake Kinneret, Israel, sampled on 6 occasions representative of different seasonal conditions was studied with scanning electron microscopy (SEM) and epifluorescence microscopy after acridine-orange staining. In near-surface (1–3 m) samples taken in October 1981 and March 1983, several unusual types of budding, appendaged, and filamentous cells were found. During lake stratification, typical large anaerobic forms (including photosynthetic green sulphur bacteria) were observed in samples from the metalimnion and deep (40 m) hypolimnion. Epifluorescence counts indicated that bacteria in the water column ranged from 0.55 to 2.67 × 10⁶ cells ml^–1.