An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy

Department of Earth Science, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
Journal of Microscopy (Impact Factor: 2.33). 10/2009; 236(1):5-10. DOI: 10.1111/j.1365-2818.2009.03207.x
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Available from: Patricia A Holden, Jan 07, 2015
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    • "After autometallograhic signal amplification, gold signals will be visible even by light microscopy [37] and the number of gold atoms should be sufficient for detection by the real-time imaging mode in NanoSIMS. Our preliminary trial using biotinylated probes and Nanogold-streptavidin [10] [19] [35] dramatically increased the gold abundance (more than 500-fold) within the cells but this method has a permeabilization problem and introduces a foreign source of additional carbon and nitrogen atoms associated with Nanogoldstreptavidin , which can alter the isotopic composition of microbial cells. Autometallographic signal amplification with Undecagoldlabeled probes can overcome these issues but it is still in the development stage because less signal amplification and higher background (low signal-to-noise ratio) have been noted. "
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    ABSTRACT: The linkage of microbial phylogenetic and metabolic analyses by combining ion imaging analysis with nano-scale secondary ion mass spectrometry (NanoSIMS) has become a powerful means of exploring the metabolic functions of environmental microorganisms. Phylogenetic identification using NanoSIMS typically involves probing by horseradish peroxidase-mediated deposition of halogenated fluorescent tyramides, which permits highly sensitive detection of specific microbial cells. However, the methods require permeabilization of target microbial cells and inactivation of endogenous peroxidase activity, and the use of halogens as the target atom is limited because of heavy background signals due to the presence of halogenated minerals in soil and sediment samples. Here, we present “Gold-ISH,” a non-halogen phylogenetic probing method in which oligonucleotide probes are directly labeled with Undecagold, an ultra-small gold nanoparticle. Undecagold-labeled probes were generated using a thiol-maleimide chemical coupling reaction and they were purified by polyacrylamide gel electrophoresis. The method was optimized with a mixture of axenic 13C-labeled Escherichia coli and Methanococcus maripaludis cells and applied to investigate sulfate-reducing bacteria in an anaerobic sludge sample. Clear gold-derived target signals were detected in microbial cells using NanoSIMS ion imaging. It was concluded that Gold-ISH can be a useful approach for metabolic studies of naturally occurring microbial ecosystems using NanoSIMS.
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    • "Recent FIB microscopes use dual-beam technology, which integrates the ion beam capability into a high-resolution scanning electron microscope. Dual-beam FIB microscopes thus allow easier localization of the area of interest, and their findings can be correlated with those from other microscopy techniques appropriate to the study of microbes, such as confocal laser scanning microscopy (e.g., Gérard et al. 2005, Ehrhardt et al. 2009), making possible additional analyses such as mapping of heavy metals (Hao et al. 2013) and 3D imaging. More details on the general procedures and examples of applications in Earth sciences can be found in de Winter et al. (2009). "
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    ABSTRACT: Microbe-mineral interactions occur in diverse modern environments, from the deep sea and subsurface rocks to soils and surface aquatic environments. They may have played a central role in the geochemical cycling of major (e.g., C, Fe, Ca, Mn, S, P) and trace (e.g., Ni, Mo, As, Cr) elements over Earth's history. Such interactions include electron transfer at the microbe-mineral interface that left traces in the rock record. Geomicrobiology consists in studying interactions at these organic-mineral interfaces in modern samples and looking for traces of past microbe-mineral interactions recorded in ancient rocks. Specific tools are required to probe these interfaces and to understand the mechanisms of interaction between microbes and minerals from the scale of the biofilm to the nanometer scale. In this review, we focus on recent advances in electron microscopy, in particular in cryoelectron microscopy, and on a panel of electrochemical and synchrotron-based methods that have recently provided new understanding and imaging of the microbe-mineral interface, ultimately opening new fields to be explored.
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    • "Direct bacterial localization would therefore require an electron-dense stain to increase the bacterial signal. There are multiple potential methods for such an electron-dense stain, the most promising of which might be the use of a gene probe labeled with a gold particle (Kenzaka et al., 2005, 2009; Ehrhardt et al., 2009). Although promising, it must be kept in mind that such methods could disturb the 3D structure of the sample. "
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    ABSTRACT: Soil habitats contain vast numbers of micro-organisms and harbour a large portion of the planet's biological diversity. Although high-throughput sequencing technologies continue to advance our appreciation of this remarkable phylogenetic and functional diversity, we still have only a rudimentary understanding of the forces that allow diverse microbial populations to coexist in soils. This conspicuous knowledge gap may be partially due the human perspective from which we tend to examine soil-borne microbes. This review focusses on the highly heterogeneous soil matrix from the vantage point of individual bacteria. Methods describing micro-scale soil habitats and their inhabitants based on sieving, dissecting, and visualizing individual soil aggregates are discussed, as are microcosm-based experiments allowing the manipulation of key soil parameters. We identify how the spatial heterogeneity of soil could influence a number of ecological interactions promoting the evolution and maintenance of bacterial diversity. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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