Observations of unstained biological specimens using a low-energy, high-resolution STEM

Low-energy, high-resolution scanning transmission electron microscopy (STEM) is introduced as a convenient method for observing unstained biological specimens. By reducing the electron energy, the cross section for light elements becomes comparable to that of conventional electron microscopy observations. The STEM mode exhibited the advantage that the induced energy loss and charge build-up in the sample affected the image to a lesser extent than in the TEM or SEM mode. Furthermore, the efficiency of an STEM detector is high, and the total radiation damage can be reduced if thermal damage due to localized heating at a slow scan operation can be overcome. We applied this method for observations of biological samples that were in the form of thin slices, fine fibers and small particles. When the supporting film for samples is absent, the resolution and the contrast of STEM images can be maintained similar to SEM and TEM images, respectively.     

Phase recovery from optical phase contrast microscopy

A procedure for phase recovery from optical phase contrast microscopy is presented. The phase and intensity corresponding to a phase contrast image, digitally acquired by a TV automated image analyzer, are reconstructed by means of a suitable iterative inversion algorithm. Applications are of major importance since they permit quantitative measurements on biological structures such as living cells.     

离散分形布朗增量随机场模型在煤微结构定量分析中的应用

本文采用离散分形布朗增量随机场（ＤＦＢＩＲ）模型对煤样微观结构的显微图像做了分形定量研究，由于分表的表面必然会产生分形的灰度图像值，故通过提取图像各像点的灰度值，计算（ＤＦＢＩＲ）场模型的分形维数，得出了不同类型煤样微结构图像的分形维数，从而为扫描电镜用于定量研究煤结构的探索了新的途径。     

Morphological and histochemical analyses of living mouse livers by new 'cryobiopsy' technique

A new 'cryobiopsy' (CB) technique has been invented for freezing the functioning livers of living mice in vivo without stopping their blood circulation. Livers of anesthetized mice were pinched off with pre-cooled CB forceps and immediately plunged into isopentane-propane cryogen. They were routinely freeze-substituted in acetone containing paraformaldehyde for light microscopy (LM) or osmium tetroxide for scanning electron microscopy (SEM). By freeze-fracturing some of them with a scalpel in liquid nitrogen before the freeze-substitution, well-preserved tissue areas were exposed only for SEM. They were either embedded in paraffin wax for LM or infiltrated with t-butyl alcohol followed by freeze-drying for SEM. Serial paraffin sections were stained with hematoxylin-eosin (HE) or histochemical periodic acid-Schiff (PAS) reaction. By HE-staining, the tissue surface areas were often compressed with the CB forceps and sinusoidal erythrocytes became aggregated side by side. In slightly deeper tissue areas, however, hepatic sinusoids were widely open with flowing erythrocytes. Lots of PAS-reaction products were well preserved in hepatocytes of the CB specimens. On the contrary, they were unevenly distributed in hepatocytes of conventionally quick-frozen specimens, and often lost in those of the conventionally dehydrated specimens. By SEM, some cell organelles, such as mitochondria and endoplasmic reticulum, and also dilated fenestrae of endothelial cells, open Disse's spaces and bile canaliculi appeared to be under normal blood circulation in the prepared CB samples. The new CB technique would be easy and useful for repeated examination of functioning organs of a living animal.     

Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284?nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2?h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.     

Photophysics of Conjugated Oligoelectrolytes Relevant to Two-Photon Fluorescence-Lifetime Imaging Microscopy

Conjugated oligoelectrolytes (COEs) comprise a class of cell-membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self-assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6-ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE-S6), and its optical properties within mammalian cells are subsequently studied. Two-photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE-S6 internalization via endocytosis. The fluorescence lifetime of COE-S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.     

非接触式扫描离子电导显微镜技术在探测活体细胞表面微结构中的应用

扫描离子电导显微镜技术是在纳米尺度进行非导电的生物样品成像的一种新型扫描探针显微镜技术。通过成功制备扫描离子电导显微镜扫描探测用纳米尺度玻璃微探针,对其进行了功能性评估;而后通过绘制探针-样品接近曲线,阐述了扫描离子电导显微镜技术实现非接触高分辨率探测的工作原理;最后采用该显微镜技术对导电标准样品及活体肾上皮A6细胞进行了表面形貌扫描成像,并与A6细胞表面形貌的扫描电镜图像进行了对照。结果表明,扫描离子电导显微镜技术不仅可实现导电样品的扫描成像,而且适宜于在生理条件下、非接触式地研究活体细胞表面的三维形貌,从而为人们深入研究细胞表面微观结构与生理功能提供了全新的技术手段。     

An energy-based three-dimensional segmentation approach for the quantitative interpretation of electron tomograms.

Electron tomography allows for the determination of the three-dimensional structures of cells and tissues at resolutions significantly higher than that which is possible with optical microscopy. Electron tomograms contain, in principle, vast amounts of information on the locations and architectures of large numbers of subcellular assemblies and organelles. The development of reliable quantitative approaches for the analysis of features in tomograms is an important problem, and a challenging prospect due to the low signal-to-noise ratios that are inherent to biological electron microscopic images. This is, in part, a consequence of the tremendous complexity of biological specimens. We report on a new method for the automated segmentation of HIV particles and selected cellular compartments in electron tomograms recorded from fixed, plastic-embedded sections derived from HIV-infected human macrophages. Individual features in the tomogram are segmented using a novel robust algorithm that finds their boundaries as global minimal surfaces in a metric space defined by image features. The optimization is carried out in a transformed spherical domain with the center an interior point of the particle of interest, providing a proper setting for the fast and accurate minimization of the segmentation energy. This method provides tools for the semi-automated detection and statistical evaluation of HIV particles at different stages of assembly in the cells and presents opportunities for correlation with biochemical markers of HIV infection. The segmentation algorithm developed here forms the basis of the automated analysis of electron tomograms and will be especially useful given the rapid increases in the rate of data acquisition. It could also enable studies of much larger data sets, such as those which might be obtained from the tomographic analysis of HIV-infected cells from studies of large populations.     

A new method combining enhanced resolution and pattern identification

We present a simple and general-purpose method able to combine high-resolution procedure with the classification and identification of objects of interest from microscopy imaging. The method is composed of two stages. First (pattern recognition), promising components (possible objects of interest) in the image are detected and small regions containing the objects of interest are extracted using a feature finder. Second, high-resolution algorithms are applied to such identified components in order to approach a multiple scales of resolution. Although the method is indeed to be applied to any microscopy technique, in this paper, we have focused the attention on biological systems, like animal cells, recorded with an atomic force microscopy.     

A Closer Look Inside Nanotubes: Pore Structure Evaluation of Anodized Alumina Templated Carbon Nanotube Membranes Through Adsorption and Permeability Studies

Although hollow nanostructures, such as nanotubes, represent a major portion of nanoscaled materials with a tremendously large application range, a detailed evaluation of their internal characteristics still remains elusive. Transmission electron microscopy is the most common analytical technique to examine the internal configuration of these structures, yet it can only provide evidence of a minimal portion of the overall material, thus, it cannot be accurately generalized. In the present paper, in addition to electron microscopy and other spot‐size analysis methods (X‐ray diffraction, Raman spectroscopy, etc.), a combination of techniques including adsorption, permeability, and relative permeability are employed in order to provide important insights into various crucial details of the overall internal surface and hollow‐space characteristics of carbon nanotube (CNT) arrays and membranes. The CNT arrays are fabricated using anodized alumina as a template in a flow‐through chemical vapor deposition (CVD) reactor. This is the first systematic approach for investigating the internal configuration of template‐based CNT arrays in detail. Key findings are made for the customized optimization of the resulting nanotube membranes for a variety of applications, including separations, nanofluidics and nanoreactors, biological capturing and purification, and controlled drug delivery and release.     

Multiparameter evanescent-wave imaging in biological fluorescencemicroscopy

Wide-field optical microscopy of live cells with nearfield precision, tracking the dynamics of subcellular organelles, imaging of single-molecule reactions with millisecond time-resolution, or watching synaptic nerve terminals "in action"-these are some examples of recent work that triggered the "renaissance" of evanescent-wave (EW) spectroscopy in biological imaging. In these studies, the goal is to markedly reduce background fluorescence from locations in the sample other than the cell/substrate interface. After a brief reminder of EW generation, we discuss how the confinement of fluorescence excitation highlights cellular structure near the plasma membrane with unprecedented detail. We then illustrate how the intensity distribution and polarization of the EW can be used to study dynamic biological processes that have neither been accessible with optical (confocal or two-photon fluorescence) nor electron microscopy, and take a glimpse of what is to come in EW imaging     

Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.     

Ultra-high-resolution scanning electron microscopy of vaccinia virus and its recombinant carrying the gag gene of human immunodeficiency virus type 1.

FL cells infected with vaccinia virus or its recombinant carrying the gag gene of human immunodeficiency virus type 1 (HIV-1) were examined by ultra-high-resolution scanning electron microscopy. Virions, whether located extracellularly or intracellularly, had a brick-shaped or watermelon appearance as a whole. Extracellular virions observed on the surface of infected cells had variable surface ultrastructures depending on the manner in which particular virions were wrapped in cell membranes. Most of the intracellular naked virions adherent to the inner face of cell surface membranes clearly exhibited ridgy, rod-shaped or globular surface structures on their surface. HIV-like particles with a diameter of about 100 nm and virions of vaccinia virus were both observed distinctly on the surface of FL cells infected with the recombinant virus.     

Composition and Structure of Oyster Adhesive Reveals Heterogeneous Materials Properties in a Biological Composite

Oyster reefs help maintain coastal ecosystems by filtering water, holding silt in place, and absorbing storm surge energy. We are just beginning to understand the chemical and structural nature of the adhesive used by these animals for building such reef communities. The adhesive has a high calcium carbonate content relative to other bioadhesives, but also appreciable levels of organics, presumably for bonding. The studies presented here use X‐ray absorption near edge structure spectroscopy, X‐ray photoemission electron microscopy, scanning electron microscopy, and microhardness methods to understand the composition, as well as the mechanical properties, of this biological material. Oyster adhesive appears to be a heterogeneous mixture of calcium carbonate and silica inclusions arranged randomly within a matrix that lacks any observable structure. Microindentation shows inclusions are significantly harder than their surroundings. This hard plus soft strategy has been noted in other biological materials, although not in any adhesives. These compositional and structural insights help propose a mechanism by which the animals generate their adhesive. Such an intriguing structure, along with resulting mechanical implications, may help explain how oyster reefs can thrive despite being subjected to demanding forces created by predators and the environment around them.     

Separation of linear and non-linear imaging components in high-resolution transmission electron microscope images

Linear and non-linear image components in high-resolution transmission electron microscope images were successfully separated by applying a bandpass filter to the three-dimensional Fourier spectrum of its through-focus series of images. In the observed lattice image of a wedgeshaped Si [110] crystal, we determined the magnitude of the contribution of the non-linear imaging components to the total image intensity distribution. The contribution was proved to become sometimes larger than that of the linear imaging component, even at a thickness of 13 nm.     

Direct observation of a stacking fault in Si(1 - x)Ge(x) semiconductors by spherical aberration-corrected TEM and conventional ADF-STEM

Spherical aberration (C(S))-corrected transmission electron microscopy (TEM) and annular dark-field scanning TEM (ADF-STEM) are applied to high-resolution observation of stacking faults in Si(1 - x)Ge(x) alloy films prepared on a Si(100) buffer layer by the chemical vapor deposition method. Both of the images clarify the individual nature of stacking faults from their directly interpretable image contrast and also by using image simulation in the case of the C(S)-corrected TEM. Positions of the atomic columns obtained in the ADF-STEM images almost agree with a projection of the theoretical model studied by Chou et al. (Phys. Rev. B 32(1985): 7979). Comparison between the C(S)-corrected TEM and ADF-STEM images shows that their resolution is at a similar level, but directly interpretable image contrast is obtained in ultrathin samples for C(S)-corrected TEM and in slightly thicker samples for ADF-STEM.     

Electron crystallography for structural and functional studies of membrane proteins

Membrane proteins are important research targets for basic biological sciences and drug design, but studies of their structure and function are considered difficult to perform. Studies of membrane structures have been greatly facilitated by technological and instrumental advancements in electron microscopy together with methodological advancements in biology. Electron crystallography is especially useful in studying the structure and function of membrane proteins. Electron crystallography is now an established method of analyzing the structures of membrane proteins in lipid bilayers, which resembles their natural biological environment. To better understand the neural system function from a structural point of view, we developed the cryo-electron microscope with a helium-cooled specimen stage, which allows for analysis of the structures of membrane proteins at a resolution higher than 3 ?. This review introduces recent instrumental advances in cryo-electron microscopy and presents some examples of structure analyses of membrane proteins, such as bacteriorhodopsin, water channels and gap junction channels. This review has two objectives: first, to provide a personal historical background to describe how we came to develop the cryo-electron microscope and second, to discuss some of the technology required for the structural analysis of membrane proteins based on cryo-electron microscopy.     

高压冷冻及冷冻替代技术在不同生物样品超微结构中的应用

高压冷冻仪在固定样品时,高压和冷冻基本是同步发生的,使得生物样品在数毫秒的时间内被瞬时固定,保证其内部结构接近于生理状态,有效减少了化学固定法中的化学试剂对细胞结构的破坏,极大程度保存了细胞在近似自然状态下的超微结构.本文分别采用化学固定法(CF)和高压冷冻固定法(HPF)对小鼠的睾丸组织、Hela细胞和酵母进行了样品...     

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.