Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells. Morphological analyses, however, have been limited because of the lack of specific protein markers and of procedures that lead to a satisfactory ultrastructural resolution. We have recently developed an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This novel approach allows excellent cell preservation and a unique resolution of the yeast morphology. Here, we present a protocol that combines this procedure with the specific labeling of the various endosomal compartments with positively charged Nanogold. In particular, we show that this new protocol generates excellent results when applied for the examination of early and late endosomes, and of mutants with an endosomal trafficking defect. Importantly, this method is compatible with immunogold labeling of protein markers, and it is consequently appropriate for localization studies of both resident and cargo proteins. This new IEM protocol will be a valuable tool for the large community of scientists using yeast as a model system to investigate the membrane transport and the biogenesis of the endosomal system. (J Histochem Cytochem 57:801–809, 2009)     

Electron tomography of amplified nanogold immunolabelling: Improvement of quality based on alignment of projections with sinograms and use of post-reconstruction deconvolution

Electron tomography of immunolabelled proteins identified with amplified nanogold particles imaged by Scanning and Transmission Electron Microscopy within thick sections is a powerful method to investigate the three-dimensional organization of complex cellular machineries. In order to increase the overall quality of the reconstructed cube, we have developed two methods that improve the tomographic reconstruction process. We first performed a very precise alignment of the projections before reconstruction with a technique using sinograms. After reconstruction, we propose to compute image restoration by calculating the Point Spread Function of the projection/back-projection system and to use it to deblur the reconstructed cubes. Improvement in the quality of the reconstructed cubes is demonstrated on images of nucleolar proteins tagged with EGFP and immunolabelled with nanogold particles.     

Formation of nanometal particles in the dialdehyde starch matrix

Environmentally friendly method of the preparation of dialdehyde starch (DAS) based composites containing nanosilver (DAS/Ag) and nanogold (DAS/Au) as reducing and protecting agents was developed. UV–vis spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction (XRD) confirmed formation of about 10 nm ball shaped Ag and Au nanoparticles situated within the polysaccharide template. Thermal properties of the composites were characterized involving differential scanning calorimetry (DSC), whereas molecular weights of polysaccharide chains of the matrix were estimated with the size exclusion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI).     

Synthesis of metallic nanoparticles using plant extracts

Biomolecules present in plant extracts can be used to reduce metal ions to nanoparticles in a single-step green synthesis process. This biogenic reduction of metal ion to base metal is quite rapid, readily conducted at room temperature and pressure, and easily scaled up. Synthesis mediated by plant extracts is environmentally benign. The reducing agents involved include the various water soluble plant metabolites (e.g. alkaloids, phenolic compounds, terpenoids) and co-enzymes. Silver (Ag) and gold (Au) nanoparticles have been the particular focus of plant-based syntheses. Extracts of a diverse range of plant species have been successfully used in making nanoparticles. In addition to plant extracts, live plants can be used for the synthesis. Here we review the methods of making nanoparticles using plant extracts. Methods of particle characterization are reviewed and potential applications of the particles in medicine are discussed.     

Direct Visualization of the Calmodulin Subunit of Phosphorylase Kinase via Electron Microscopy Following Subunit Exchange

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.     

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)‐modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA–As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)–ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8–230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Reprint of "On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography" [J. Struct. Biol. 159 (2007) 507-522

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

Amperometric immunosensor based on multiwalled carbon nanotubes/Prussian blue/nanogold-modified electrode for determination of α-fetoprotein

In this article, a conspicuously simple and highly sensitive amperometric immunosensor based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrode (GCE) surface is proposed for the detection of α-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proven to show much better electrochemical stability and a larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecules but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg/ml (at 3δ), and the linear working range spanned the concentrations of AFP from 0.01 to 300 ng/ml. Moreover, the immunosensor, as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significant, the assay mentioned here is simpler than the traditional enzyme-linked immunosorbent assay (ELISA), and an excellent correlation of levels of AFP measured was obtained, indicating that the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis.