Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

冷冻聚焦离子束-扫描电镜成像技术研究进展

冷冻聚焦离子束-扫描电镜成像(Cryo-FIB-SEM)是一种新兴的成像检测技术,在原位进行冷冻聚焦离子束切割和冷冻扫描电镜成像,为研究天然含水状态下生物样品内部未被破坏的原始结构打开了一扇窗口。近年来,该技术在生命科学领域的应用研究取得了一系列重要进展。该文对其在冷冻体积连续成像、冷冻光电关联成像、冷冻透射扫描成像、冷冻含水切片制备监控及冷冻扫描图像处理等方面的研究进展进行综述,并展望了该技术在大体积生物样品三维原位成像研究领域的前沿性发展趋势,以期推动Cryo-FIB-SEM技术在生物样品三维结构研究中的应用。     

High-resolution imaging of the microbial cell surface

Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of cell-drug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.     

Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes.     

A nano-view of West Nile virus-induced cellular changes during infection

Background  

Microscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope's resolution or the invasive nature of the sample preparation procedures. It is also difficult to have a technique that would allow simultaneous imaging of both surface and sub-surface on the same cell. This has hampered endeavours to elucidate virus-host interactions. Atomic Force Microscopy (AFM), which is commonly used in the physical sciences, is now becoming a good correlative form of microscopy used to complement existing optical, confocal and electron microscopy for biological applications     

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

Summary Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.Abbreviations EPS extracellular polysaccharide - EM electron microscopy - LTSEM low temperature scanning electron microscopy - SEM scanning electron microscopy - TEM transmission electron microscopy - TSA tryptic soy agar - TSB tryptic soy broth Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells,tissues, and organs

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack a direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution three-dimensional (3D) volumes of intact plant samples from the cell to the whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High-quality 3D volume data from our enhanced methods facilitate sophisticated and effective computational segmentation. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high-resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.

Lab-based X-ray microscopy allows high-resolution 3D imaging of intact plant samples over a wide range of sample types and sizes, filling the imaging gap between light and electron microscopy.     

Seeing is believing: novel imaging techniques help clarify microbial nanowire structure and function

Novel imaging approaches have recently helped to clarify the properties of ‘microbial nanowires’. Geobacter sulfurreducens pili are actual wires. They possess metallic‐like conductivity, which can be attributed to overlapping pi‐pi orbitals of key aromatic amino acids. Electrostatic force microscopy recently confirmed charge propagation along the pili, in a manner similar to carbon nanotubes. The pili are essential for long‐range electron transport to insoluble electron acceptors and interspecies electron transfer. Previous claims that Shewanella oneidensis also produce conductive pili have recently been recanted, based on novel live‐imaging studies. The putative pili are, in fact, long extensions of the cytochrome‐rich outer membrane and periplasm that, when dried, collapse to form filaments with dimensions similar to pili. It has yet to be demonstrated whether the cytochrome‐to‐cytochrome electron hopping documented in the dried membrane extensions takes place in intact hydrated membrane extensions or whether the membrane extensions enhance electron transport to insoluble electron acceptors such as Fe(III) oxides or electrodes. These findings demonstrate that G. sulfurreducens conductive pili and the outer membrane extensions of S. oneidensis are fundamentally different in composition, mechanism of electron transport and physiological role. New methods for evaluating filament conductivity will facilitate screening the microbial world for nanowires and elucidating their function.     

Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe³⁺, Cu²⁺, Zn²⁺, and Hg²⁺, illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems.     

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.     

Utilization of hydrogen,acetate, and “noncompetitive”; substrates by methanogenic bacteria in marine sediments

We have used a relatively new microscopical technique, environmental scanning electron microscopy (ESEM), along with transmission electron microscopy (TEM) and light microscopy, to investigate a unique microbial community from a temperate-climate, cold sulfide spring near Ancaster, Ontario, Canada. ESEM allows the viewing of fully hydrated specimens that have not undergone the structural or chemical alterations imposed by the extensive procedures necessary for viewing biological specimens in a vacuum. Besides allowing visualization of microorganisms in their natural form and as intact assemblages, ESEM also detects elements, especially those lighter than Si, which tend to be lost or masked by the processes used to prepare samples for conventional SEM and for TEM thin sections. In this study we report new information about the structure of bacteriogenic sulfur deposits and their relationship to the structural aspects of a natural microbial community from a cold sulfide spring.     

一株促甲烷氧化假单胞菌Pseudomonas putida P7的分离及电活性特征

微生物胞外呼吸是厌氧环境中控制性能量代谢方式,直接驱动着C、N、S、Fe等关键元素的生物地球化学循环。微生物纳米导线（Microbial nanowires）的发现,被认为是微生物胞外呼吸的里程碑事件,推动了电微生物学（Electromicrobiology）的形成与发展。微生物纳米导线是一类由微生物合成的,具有导电性的纤维状表面附属结构。通过细菌纳米导线,微生物胞内代谢产生的电子可以长距离输送到胞外受体或其他微生物,改变了电子传递链仅仅局限于细胞胞内的认识,从而大大拓展了微生物-胞外环境互作的范围。微生物纳米导线的良好导电性,赋予了其作为天然纳米材料的广阔应用前景。目前,微生物纳米导线的导电机制、生态功能及其在生物材料、生物能源、生物修复及人体健康多领域的应用,已经成为新兴电微生物学的前沿与热点。然而,微生物纳米导线的生物学、生态学功能尚不清楚,它的电子传递机制仍存在分歧。本文在系统性总结微生物纳米导线性质、功能的基础上,以Geobacter sulfurreducens和Shewanella oneidensis纳米导线为模型,详细阐述了纳米导线的组成与结构、表征与测量方法、导电理论（类金属导电学说与电子跃迁学说）及其潜在的应用,最后提出了未来微生物纳米导线研究的重点方向、挑战与机遇。     

Imaging vesicular dispersions with cold-stage electron microscopy

A fast-freeze, cold-stage transmission electron microscopy technique which can incorporate in situ freeze-drying of the sample is described. Its use in elucidating structure in unstained and stained, hydrated and freeze-dried, aqueous vesicular dispersions of biological and chemical interest is demonstrated with vesicles of l-α-phosphatidylcholine (bovine phosphatidylcholine) and of the synthetic surfactant sodium 4-(1′-heptylnonyl)benzenesulfonate (SHBS). The contrast features observed in transmission electron microscope images of frozen, hydrated samples are identified and explained with the dynamical theory of electron diffraction. Radiolysis by the electron beam is shown to increase contrast in vesicle images and to change their structure and size. Micrographs illustrate the freeze-drying of a dispersion in the microscope; the process causes vesicles to shrink and collapse.     

Stay connected: Electrical conductivity of microbial aggregates

The discovery of direct extracellular electron transfer offers an alternative to the traditional understanding of diffusional electron exchange via small molecules. The establishment of electronic connections between electron donors and acceptors in microbial communities is critical to electron transfer via electrical currents. These connections are facilitated through conductivity associated with various microbial aggregates. However, examination of conductivity in microbial samples is still in its relative infancy and conceptual models in terms of conductive mechanisms are still being developed and debated. The present review summarizes the fundamental understanding of electrical conductivity in microbial aggregates (e.g. biofilms, granules, consortia, and multicellular filaments) highlighting recent findings and key discoveries. A greater understanding of electrical conductivity in microbial aggregates could facilitate the survey for additional microbial communities that rely on direct extracellular electron transfer for survival, inform rational design towards the aggregates-based production of bioenergy/bioproducts, and inspire the construction of new synthetic conductive polymers.     

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley-Liss, Inc.     

Cryo-electron microscopy of vitreous sections of native biological cells and tissues

Cryo-electron microscopy of vitreous sections (CEMOVIS) is, in principle, the ultimate method of specimen preparation. It consists in ultra-rapid cooling of a sizable sample of biological material that is cut into thin sections. These are subsequently observed at low temperature in their fully hydrated vitreous state. Here, we show that CEMOVIS reveals the native state of cells and tissues with unprecedented quality and resolution. What is seen differs considerably from what conventional electron microscopy has shown previously and it is seen with more details. Our findings are demonstrated with images of cyanobacteria and skin.     

Towards native-state imaging in biological context in the electron microscope

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.     

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.