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N A N O P R O B E S     E - N E W S

Vol. 9, No. 7          July 31, 2008


Updated: July 31, 2008

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Correlative and Dual Labeling with FluoroNanogold

FluoroNanogold is a unique combined fluorescent and gold labeled immunoprobe: it is the only immunoprobe available that can provide both fluorescent and gold labeling with a single probe and a single labeling procedure. These probes contain and antibody Fab' fragment covalently linked to both the 1.4 nm Nanogold® label and a fluorescent label (currently, a choice of Alexa Fluor®* 488 or 594, or fluorescein, are available; other fluorescent labels are planned). The covalent linkage assures stability, and because these probes do not require stabilization with additional macromolecules, they are smaller than IgG molecules and show the same high penetration and antigen access of Nanogold-Fab' fragments.

These unique probes may be used for a number of different applications and combinations of different microscopic methods:

  • Correlative fluorescence and transmission electron microscopic labeling
  • Checking labeling by fluorescence before processing for electron microscopy
  • Combined fluorescence and scanning electron microscopy
  • Correlative optical and X-ray fluorescence microscopy

Correlative fluorescence and transmission electron microscopic labeling

Our Alexa Fluor®* FluoroNanogold probes offer superior fluorescence labeling performance:

  • Increased fluorescence brightness and higher quantum yield.
  • Improved solubility: lower background signal and higher signal-to-noise ratios.
  • Fluorescence remains high and consistent across a wider pH range.

Since we now offer both Alexa Fluor®* 488 and Alexa Fluor®* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.

John Robinson and Toshihiro Takizawa pioneered the uses and applications of combined fluorescent and gold probes, and their studies on the distribution of caveolin-1 in ultrathin cryosections of terminal villi of the human term placenta by fluorescence and immunoelectron microscopy of ultrathin cryosections provides an example of correlative methodology. Their procedure enables high spatial resolution by fluorescence microscopy because there is essentially no out-of-focus fluorescence. Electron microscopic immunolabeling obtained with conventional colloidal gold and FluoroNanogold were compared using a particle counting procedure: a higher number of particles was found with silver-enhanced FluoroNanogold than with colloidal gold.

[Alexa Fluor 594 FluoroNanogold: correlative fluorescence and TEM labeling (63k)]

Correlative fluorescence and electron microscopic labeling with Alexa Fluor 594-Streptavidin. Localization of caveolin-1a in ultrathin cryosection of human placenta; caveolin 1 alpha is primarily located to caveolae in placental endothelial cells. One-to-one correspondence is found between fluorescent spots (upper right) and caveola labeled with gold particles (lower right). Ultrathin cryosections, collected on formvar film-coated nickel EM grids, were incubated with chicken anti-human caveolin-1a IgY for 30 minutes at 37°C, then with biotinylated goat anti-chicken F(ab')2 (13 µg/mL, 30 minutes at 37°C), then with Alexa Fluor 594 FluoroNanogold-Streptavidin (1:50 dilution, 30 minutes at room temperature). Non-specific sites on cryosections were blocked with 1% milk - 5% fetal bovine serum-PBS for 30 minutes at room temperature (figure courtesy of T. Takizawa, Ohio State University, Columbus, OH).

Tissue was cut into small pieces and fixed in freshly prepared 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 2 hr at room temperature. The samples were washed and embedded in 10% gelatin in the same buffer. The solidified gelatin was cut into smaller pieces and then cryoprotected by infiltration with 2.3 M sucrose in 0.1 M sodium cacodylate (pH 7.4) overnight at 4°C. Ultrathin cryosections (100-nm thickness or less) were cut on a Cryo P diamond knife and collected on droplets of 0.75% gelatin2.0 M sucrose or 1 % methylcellulose-1.15 M sucrose, then transferred to nickel Maxtaform finder grids to facilitate location of specific structures when going from the optical to the electron microscope. Pick-up solutions contained 0.05% sodium azide so that the cryosections could be stored until needed. Sections were immersed in a solution containing 1% non-fat dry milk and 5% fetal bovine serum in PBS (MFBSPBS) for 15 min at 37°C to remove the sucrose and gelatin, then washed three times in PBS and incubated in MFBSPBS with 0.05 % sodium azide to block nonspecific protein binding sites.

The grids were incubated with biotin-labeled goat anti-chicken (13 g/ml in MFBSPBS) for 30 min at 37°C, washed in PBS for 12 min with four changes, immersed in MFBSPBS, then incubated with Alexa Fluor 594 FluoroNanogold-streptavidin (diluted 1:50 in MFBSPBS) for 30 min at room temperature. The grids were then washed in PBS for 15 minutes with five changes and mounted on a glass microscope slide in PBS containing 1% N -propyl gallate and 50% glycerol, pH 8.0, to retard photobleaching, overlaid with an 18-mm round glass coverslip. The cryosections were examined immediately by optical microscopy and images were collected. The locations of regions of interest on the finder grid were noted for relocation in the electron microscope. The temporary slide preparations were then disassembled and the grids washed in PBS with five changes over 15 min. The ultrathin cryosections were then fixed in 2% glutaraldehyde in PBS for 30 min and washed in distilled water for 6 min with four changes. The sides of the grids opposite the sections were dried with filter paper. The grids were then floated on drops of distilled water and subsequently on drops of 50 mM 2-[N-morpholino] ethanesulfonic acid buffer, pH 6.15, for 4 minutes with two changes. FluoroNanogold bound to the cryosections was silver enhanced for 3 minutes to render them visible in the sections; a positive contrast enhancement procedure was used to visualize membrane profiles in the ultrathin cryosections. The same regions examined by fluorescence microscopy were relocated and electron micrographs collected.

Reference:

  • Takizawa, T., and Robinson, J. M.: Ultrathin Cryosections. An important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem., 51, 707-714 (2003).

Check labeling by fluorescence before EM processing

FluoroNanogold can also be used to check labeling by fluorescence in order to select samples to process for EM. For example, Iliev and co-workers recently used FluoroNanogold as part of their ultrastructural investigations into the effects of modifications to the amino acid sequence motifs in the microtubule-binding repeats of tau, a pathological marker of Alzheimers disease (AD) and other tauopathies.

Reference:

  • Iliev, A. I.; Ganesan, S.; Bunt, G., and Wouters, F. S.: Removal of pattern-breaking sequences in microtubule binding repeats produces instantaneous tau aggregation and toxicity. J. Biol. Chem., 281, 37195-37204 (2006).

Correlative fluorescence and scanning electron microscopic labeling

Elizabeth Schröder-Reiter and group used FluoroNanogold, visualized by fluorescence and high-resolution 3D analytical SEM, to show the architecture and DNA distribution a nucleolus organizing region (NOR) with atypical peg-like terminal constriction on metaphase plant chromosomes. In their current paper, which appears in a special issue of Methods in Cell Biology dedicated to the biological applications of electron microscopy, they discuss improvements in signal localization, labeling efficiency, and structural preservation in the SEM ISH procedure that have allowed 3D SEM analysis of the NOR structure and rDNA distribution for the first time. High-resolution 3D analytical SEM showed that the architecture and DNA distribution of the peg-like NOR were typical for chromosomes, although the chromomeres were significantly smaller. Improvements to the SEM ISH procedure enabled more accurate signal localization, higher labeling efficiency, and better structural preservation, and for the first time this allowed 3D SEM analysis of the peg-like NOR structure and rDNA distribution. FluoroNanogold proved to be an attractive tool that allows efficient immunodetection in both LM and SEM. Based on the data obtained, a model was proposed for the peg structure and its mode of condensation.

[Alexa Fluor 488 FluoroNanogold-Fab' and results with it (87k)]

Left: Structure of Alexa Fluor®* 488 FluoroNanogold - Fab' and Streptavidin, showing covalent attachment of components. Center: Fluorescent staining obtained using Alexa Fluor 488 FluoroNanogold as a tertiary probe to label red blood cells. Specimen is a slide from the NOVA Lite ANA HEp-2 test, an indirect immunofluorescent test system for screening anti-nuclear antibodies in human serum, stained using positive pattern control human sera, a Mouse anti-Human secondary antibody, and Alexa Fluor 488 FluoroNanogold tertiary probe. Specimens were washed (PBS, 30 minutes) between each step, then blocked by addition of 7% nonfat dried milk to the tertiary antibody solution (original magnification x 400). Right: Scanning electron micrograph of a peg-like terminal constriction of an Oziroë biflora (plant, Hyacinthaceae) chromosome. The image shows both chromosome topography (secondary electron signal) and hybridized enhanced gold signals (superimposed back-scattered electron signals, yellow) labeling 45S rDNA in the nucleolus organizing region with Alexa Fluor®* 488 FluoroNanogold-Streptavidin (micrograph courtesy of Elizabeth Schröder-Reiter and Gerhard Wanner)

For in situ hybridization, a plasmid VER17 encoding part of the 18S, the 5.8S, most of the 25S, and the internal transcribed spacers of Vicia faba 45S rRNA, was used as an rDNA-specific probe. 45S rDNA was labeled by nick 51 translation with biotin-16-dUTP, and for ISH, 20 ng probe was applied per slide. For correlative LM and SEM ISH, the procedure was shortened by omitting the enzymatic (pepsin, RNase) treatment and intermediate dehydration steps, and to ensure structural preservation of the chromosomes, it was essential that all air-drying steps were avoided. Prior to detection of biotin-labeled probes, slides were incubated in a blocking solution (5% bovine serum albumin in 4 standard sodium citrate (SSC) with 0.2% Tween-20) for 30 minutes at 37°C. Alexa Fluor®* 488 FluoroNanogoldstreptavidin, diluted at 1:100 in 1% bovine serum albumin in 4 SSC with 0.1% Tween 20), or fluorescein isothiocyanate (FITC)streptavidin was then applied, incubated for 1 hour in a moistened, light-protected chamber at 37°C, and washed 35 min in 2 SSC. Specimens were mounted in 1% (w/v) 4',6-diamidino-2-phenylindole (DAPI) dissolved in Vectashield (Vector, Burlingame, CA, USA), slides examined, and fluorescent images digitally recorded.

After specimens were evaluated by fluorescence microscopy, coverslips were removed by floating in 100% ethanol (EtOH). Specimens were washed three times with 100% ethanol to remove the Vectashield, washed with distilled water, and silver-enhanced for 5 minutes using HQ Silver. After a final wash series in distilled water, slides were washed in 100% acetone, critical point dried from CO2, checked by phase contrast light microscopy, cut to size with a glass cutter, and mounted with double-sided tape to an aluminum stub with conductive carbon cement. Unlabeled specimens were sputter coated with platinum to a layer of approximately 35 nm and examined at accelerating voltages between 8 and 15 kV; labeled specimens (with Nanogold or with Pt blue) were carbon-coated by evaporation to a layer of 35 nm and examined at either 25 or 30 kV. Backscattered electrons (BSE) were detected with a YAG-type detector (Autrata). Secondary electron (SE) and BSE images were recorded simultaneously.

References:

  • Wanner, G., and Schröder-Reiter, E.: Scanning electron microscopy of chromosomes. Methods Cell Biol., 88, 451-474 (2008).

  • Schröder-Reiter, E.; Houben, A.; Grau, J, and Wanner, G.: Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy. Chromosoma, 115, 50-59. (2006).

Correlative optical and X-ray fluorescence microscopy Synchrotron-based X-ray fluorescence microscopy (microXRF) provides high-resolution localization of a wide range of biologically relevant elements, including gold and other metals, in tissues and cells. When combined with fluorescence microscopy, it enables correlation of the cellular distribution of a target with the ultrastructural mapping of many different elements, raising the possibility of ultrastructural labeling of multiple sites distinguished by different elemental labels in a similar manner to that described previously using electron spectroscopic imaging. This method provided two-dimensional maps with submicron resolution for gold, as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the gold-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

McRae and group used FluoroNanogold for correlative fluorescence and micro X-ray fluorescence microscopy. MicroXRF elemental maps with well-defined subcellular resolution were obtained by growing NIH 3T3 mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids. The cells were fixed at room temperature for 30 minutes with pre-warmed (37°C) 3.7% paraformaldehyde freshly prepared in phosphate-buffered saline (PBS; pH 7.2), permeabilized with 0.2% Triton X-100 in PBS (pH 7.2) for 10 minutes, blocked for one hour, then incubated with either mouse anti-GS28 IgG1 (cis-Golgi marker; 1:300 dilution), or anti-OxPhos complex V IgG1 (mitochondrial marker; 1:300 dilution) for one hour. After washing thoroughly with 0.05% Tween-20 in PBS, they were incubated with Alexa Fluor 488 FluoroNanogold anti-mouse secondary antibody (1:10 dilution) for one hour, then washed again with Tween-20. Optical fluorescence micrographs were acquired by mounting the silicon nitride windows onto slides, using PBS as mounting medium, and imaging with an inverted fluorescence microscope equipped with a standard filter set (FITC).

Immediately after fluorescence imaging, the cells were rinsed quickly with PBS then twice with isotonic ammonium acetate (0.1M) in ultrapure water, air-dried overnight, then examined by scanning X-ray fluorescence microscopy at the 2-IDD beamline of the Advanced Photon Source at Argonne National Laboratory (IL, USA). The grids were placed onto a kinematic specimen holder suitable for both optical and X-ray fluorescence microscopy. Target cells imaged previously by standard fluorescence microscopy were located on the grid using a high spatial resolution motorized x/y stage. A Fresnel zone plate was used to focus the monochromatic X-ray beam from an undulator source to a spot size of 0.2 x 0.2 µm2 on the specimen, and an incident photon energy of 11.95keV was chosen to ensure excitation of the Lalpha line of gold. The sample was raster scanned through the beam at 298K under a helium atmosphere. Pixel step size was set to 0.2 µm and the entire X-ray spectrum recorded at each pixel using an energy dispersive germanium detector. Elemental maps were created by spectral filtering, using spectral regions of interest matched to characteristic X-ray emission lines to determine the fluorescence signal for each element.

Reference:

  • McRae, R.; Lai, B.; Vogt, S., and Fahrni, C. J.: Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles. J. Struct. Biol., 155, 22-29 (2006).

More information:

* Alexa Fluor is a registered trademark of Invitrogen (Molecular Probes), Inc.

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How to Gold-Label Peptides

Peptides are one type of molecule that are very difficult to label with conventional colloidal gold, but relatively easy to label with Nanogold®. Gold-labeled peptides are novel and useful probes; because the Nanogold® particle is much smaller than an antibody, Nanogold-labeled peptides are much smaller even than Fab' conjugates, and their small size in particular gives them several advantages for labeling:

  • Restricted targets are accessed more easily by small peptide probes.
  • Smaller probes mean higher resolution - the gold label is closer to the target.
  • Labeling is more simple - no cross-reactivity issues with secondary probes.
Segond Von Banchet and Heppelmann published one of the first such studies, using labeled substance P (SP), an 11 amino acid residue peptide, to demonstrate the distribution of its binding sites in histological sections of rat spinal cord. The peptide was labeled at the primary amino group with using Mono-Sulfo-NHS-Nanogold. One µmol of SP was dissolved in 500 µL of HEPES (20 mM, pH 7.4), then added to a 500 µL solution of 30 nmol Mono-Sulfo-NHS-Nanogold dissolved in double-distilled water, and incubated one hour at room temperature (RT); the reagent was added to an excess of the peptide. Labeled peptide conjugate was separated from unbound peptides using membrane centrifugation (Amicon Centricon-10 system) with a molecular weight cut-off of 10 kDa, spinning at 14.000 x g for 65 min at 4°C. The filter carrying the labeled peptides was placed upside down into a new vial and spun again for 3 minutes at 1000 x g and 4°C to collect the peptide-gold conjugate. This procedure was repeated twice to remove all unbound peptide. The peptide-gold conjugates were diluted in 10 mM sodium phosphate-buffered saline (PBS; 150 mmol NaCl, pH 7.4) containing 10 mM sodium azide, 1% bovine serum albumin BSA; w/v), leupeptin (4 µg/mL), and 2 M sucrose, aliquottcd into 100 µL tubes, and stored at -20°C.

[Nanogold labeling of Substance P (39k)]

(Left) Labeling of the 11-residue peptide Substance P (SP) using Mono-Sulfo-NHS-Nanogold®. (Right) SDS-PAGE of Proteins of the rat spinal cord (a) India ink stained, showing many proteins; (b) 125I-SP staining, showing binding to 58 and 38 kDa target protein bands. (c) 125I-SP and excess SP, showing SP competes for labeled protein binding. (d) SP-Nanogold (silver enhanced) showing binding to 58 and 38 kDa bands, similar to 125I-SP (lane b). (e) SP-Nanogold and excess SP, showing that excess SP competes off labeled protein. This shows that the Nanogold- labeled 11-mer peptide Substance P retains its native binding properties.

In Western blots of membrane fractions of rat spinal cord, specific binding occurred at 38 and 58 kDa. This binding was competitively suppressed by adding the native peptide. In addition, the SP-Nanogold conjugate was able to displace the corresponding 125I-labeled peptide from binding proteins. In histological sections, binding sites could were shown in various parts of rat brain and spinal cord with distribution patterns comparable to those found by autoradiographic methods. Adding the native peptide or a neurokinin 1 receptor agonist markedly reduced labeling of the tissue, whereas only a slight reduction was obtained after adding neurokinin A. These results show that labeling SP with Nanogold, even though the Nanogold particle is considerably larger than SP, resulted in an effective probe in which the binding properties of native SP are preserved which could be used to demonstrate its binding sites. This new labeling method combines the advantages of receptor-ligand affinity binding with a non-radioactive detection system, and the authors showed that can be used for labeling peptides in general, thus offering an alternative or addition to other methods used in study of membrane receptors.

Another class of peptides are naturally occurring venoms - neurotoxins with very precise and potent effects. Because they are small molecules, they are also excellent probes for their sites of action. Alpha 7 subunit-containing nicotinic acetylcholine receptors (alpha 7* nAChR) plays a role in a variety of processes in the mammalian brain, and identifying the precise cellular distribution of alpha 7* nAChRs relative to the local neurochemical environment is necessary in order to understand these roles. Alpha-bungarotoxin, a potent snake venom which is actually an 8,000 MW, 74 amino acid peptide, has a unique advantage as a probe because it only binds to assembled alpha 7* nAChRs. To take advantage of this binding, Jones and group labeled this peptide with Mono-Sulfo-NHS-Nanogold, using the standard procedure given in our product instructions, and the product was purified by gel filtration. Displacement assays showed that alpha-Bgt binding was preserved in the Nanogold conjugate: Ki values are 7.8 and 4.2 nM for gold conjugated alpha-Bgt and unconjugated alpha-Bgt, respectively.

The authors then used the labeled peptide to stain alpha 7* nAChR sites using a pre-embedding labeling procedure and observation both by light and electron microscopy. Regions of interest were dissected and tissue blocks glued to the stage of a vibrating microtome, submerged in ice cold artificial cerebrospinal fluid (ACSF). At the light microscope level, labeled binding sites were mostly on the section surface, associated with the cell body and dendritic membranes. By electron microscopy, gold alpha-Bgt binding was observed on, or within a few micrometers, of the section surface. Labeling in the stratum radiatum was observed on dendritic membranes at both synaptic and perisynaptic loci; no cytoplasmic labeling was observed. Sections were transferred to glass vials containing cold ACSF on a shaker, allowed to reach room temperature, washed 3 x 5 minutes in ACSF and then blocked in ACSF with 0.2 % acetylated bovine serum albumin (ACSF-BSAc) for 30 minutes. After rinsing briefly in ACSF, sections were incubated in gold alpha-Bgt (1100 nM) in ACSF-BSAc for 1 h, then washed 3 x 10 minutes in ACSF-BSAc and fixed in ACSF containing 2.5 % glutaraldehyde for 30 minutes. Fixed sections were washed 4 x 10 minutes in deionized water (DW) and silver enhanced for 20 minutes (for light microscopy; SE-LM reagent, Aurion) or one hour (for electron microscopy; SE-EM reagent, Aurion). Sections for light microscopy were air-dried onto poly-l-lysine coated microscope slides, defatted by passing through an alcohol series, Nissl stained, dehydrated and mounted in DPX mountant. For electron microscopy, sections were dehydrated through an alcohol series into propylene oxide, infiltrated with epoxy resin overnight, flat embedded on microscope slides then cured in an oven at 60°C for 48 hours. Ultrathin sections (60 nm) were collected onto pioloform-coated nickel slot grids and counterstained with uranyl acetate and lead citrate. At the light microscope level, labeled binding sites were mostly on the section surface, associated with the cell body and dendritic membranes. By electron microscopy, gold alpha-Bgt binding was observed on, or within a few micrometers, of the section surface. Labeling in the stratum radiatum was observed on dendritic membranes at both synaptic and perisynaptic loci; no cytoplasmic labeling was observed.

References:

  • Schmidt, K.; Segond von Banchet, G., and Heppelmann, B.: Labelling of peptides with 1.4-nm gold particles to demonstrate their binding sites in the rat spinal cord. J. Neurosci. Methods., 87, 195-200 (1999).

  • Segond von Banchet, G., and Heppelmann, B.: Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles. J. Histochem. Cytochem., 43, 821-827 (1995).

  • Jones; I. W.; Barik, J.; O' Neill, M. J., and Wonnacott, S.: Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors. J. Neurosci. Methods, 134, 65-74 (2004).

Peptides can present different issues to antibodies and larger proteins when conducting labeling reactions. Some of the issues that can arise include:

  • Precipitation and aggregation: some peptides have low aqueous solubility; because the Nanogold label is quite large, the properties of others can change dramatically upon conjugation. Picking a buffer that matches the solubility requirements of your peptide can be helpful in this case: if it requires a high ionic strength, this should not be a problem provided that the pH is kept within the labeling range. In some cases, conducting the reaction in a proportion of an organic solvent such as isopropanol or acetonitrile also helps solution. A useful buffer is triethylammonium bicarbonate, prepared by bubbling carbon dioxide through water / triethylamine: this is a effective solubilizer for Nanogold and may help pull conjugates into solution, and is compatible with many chromatography gels.

  • Separation methods: because of their small size, peptide conjugates usually require different separation procedures to larger antibody or protein conjugates. Separation is usually addressed using reaction stoichiometry. A general strategy is to ensure that the reagent which is closest to the labeled product in size is the limiting reagent, so that in the conjugation reaction, it is used up to leave an excess of the reagent which is more different in size and hence more easily separated. Chromatography media should be selected carefully: this is discussed, with a gel selection guide, in a previous article.

  • Conjugate concentrations: The presence of the Nanogold, because it is large relative to many peptides, can make the concentration of active peptide appear greater than it is. Concentrations should be converted to molecular or molar concentrations for comparison, rather than weight or mass per volume.

More information:

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Gold Nanoparticles: Nanotechnology for Medical Imaging

Gold nanoparticles make excellent X-ray contrast agents, and we have used them for applications such as angiography and in vivo vascular casting. AuroVist is our first commercial application of this technology: it is a gold nanoparticle contrast agent for use in research applications with small animals, in particular microCT and CT imaging. AuroVist produces contrast enhancement up to ten times that of iodine reagents, providing high-resolution, high-contrast images of blood vessels, organs, other anatomical structures and tumors in animals. It is highly soluble, biocompatible, and stable to the vascular and tissue environment.

AuroVist mouse, kidney and inferior vena cava images [(104k)]

(Upper left): Live mouse, 5 minutes after injection. (Upper right) Imaging of kidney in live mouse 1 hour after injection, showing kidney contrast and fine structure (bar = 1 mm). (Lower left): Live mouse, 2 minutes after injection showing vascular fine structure (bar = 5 mm); (Lower right) MicroCT of mouse inferior vena cava (bar = 1 mm).

AuroVist is a stabilized 1.9 nm gold particle. It provides better contrast than iodine for both micro-CT and clinical CT applications.* At appropriate beam energies, gold achieves a contrast up to three times greater than iodine per unit mass, yielding initial blood contrast greater than 500 Hounsfeld Units (HU). Gold concentrations up to four times those of iodine can be achieved, providing a total contrast gain of up to ten times or more. In addition, AuroVist gives other enhanced performance features:

  • Clears through kidneys, making it ideally suited to kidney imaging. Kidney fine structure may be visualized up to an hour or more after injection; concentration in the kidneys can provide contrast values as high as 1,500 HU or more.

  • Longer blood residence time than iodine agents, due to its larger size (1.9 nm gold core, ~50,000 Da).

  • High contrast (>500 HU initial in blood, kidneys >1,500 HU).

  • Permeates angiogenic endothelium, enabling imaging of tumors.

  • Concentration >4 times that of standard iodine agents (up to 1.5 g Au/cc).

  • Can be imaged using standard microCT.
  • Low toxicity (LD50 >1.4 g Au/kg).

  • Up to 10 times the contrast of standard iodine agents (Gold absorbs ~3 times more than an equivalent weight of iodine at 20 and 100 keV and can be ~4 times more concentrated, giving more than 10-fold combined contrast enhancement.

  • Low osmolality, even at high concentrations

  • Low viscosity, similar to water; easy to inject, even into small vessels.

  • Yields enhanced radiotherapy dose.

AuroVist is a new product, and therefore it has not been optimized in all possible applications. However, the following guidelines may be helpful in obtaining the best results.

Best instrument and beam settings

The absorption increases by a significant factor (jump ratio) above the gold L and K edges. The X-ray properties of gold, showing the jump ratios for these regions, are shown in the table below. It is therefore advantageous to image using these absorptions. The settings below are appropriate for the different instruments.

  Energy (keV) µ/p(cm2/g) jump ratio
  11.8 75.8  
L3 11.9 187.0 2.5
  13.6 128.3  
L2 13.7 176.4 1.4
  14.3 158.8  
L1 14.4 183.0 1.2
  80.6 2.1  
K 80.7 8.9 4.2
  • Mammography: These instruments are suitable for small animal imaging. Use of lower kVp (e.g., 22 kVp) is recommended to take advantage of the L edge Au absorptions. Exposures are typically 1 second or less for a mouse, so live imaging is possible. Resolution can be < 0.1 mm.

  • Clinical CT: 80 kVp gives the greatest attenuation, but higher voltages, particularly with filtering can make use of the Au K edge; beam energy can be tuned to just above gold's 80.7-keV K-edge. Imaging time is typically a few seconds, with resolution ~0.3 mm.

  • MicroCT: Here the resolution is increased (to even 2 microns), but the tube power is typically ~100 times less than a clinical unit. Fine area 2D detectors mean that many tiny pixels must each receive enough counts. This then requires a much longer imaging time (e.g., 0.5 - 2 hours) than clinical CT. Many units also slow the tube rotation such that only 1 revolution is done in the selected imaging time (e.g., 1 hour). Animal movement must be minimized during this time. One solution is to sacrifice the animal, but live imaging has been accomplished if the region can be gated or immobilized during the imaging time. Beam energy should be just above golds 12-14-keV L-edge.

How to minimize toxicity

Certain strains of mice appear to be more tolerant of AuroVist. For Balb/C, the LD50 is ~ 3.2 g Au/kg. Nude mice and C3H mice also seem to respond about the same. Some outbred mice, however, appear to have a lower LD50 of about > 1.4 g Au/kg. If you are using outbred mice, or a different strain to those mentioned above, it is recommended that you use this lower value. The following suggestions may be helpful:

  • Start with a moderate dose, such as 120 or 160 mg/mL. The value of 270 mg/mL reported in our paper in the British Journal of Radiology was the highest value tested; at high concentrations acute toxicities increase rapidly. A modest reduction in dose from these levels can significantly reduce toxicity without compromising your results.

  • Centrifuge and then filter the reconstituted reagent through a sterile filter immediately before injection to ensure that no aggregates or larger particles are present. Larger particles and aggregates can impact biocompatibility, and may also have reduced stability and hence a higher tendency to deposit in tissues and organs, with negative consequences.

  • Use one of the pure-bred mouse strains mentioned above, which are known to have higher tolerance. If you are planning to conduct multiple experiments using a different strain, it may be worthwhile to test your strain first.

We encourage you to tell us how well it works in your application, or if you encounter problems; that way, you can help contribute to the knowledge base on this reagent and its applications.

We have received several inquiries about whether a targeted version of this reagent is available. While the preparation of a targeted gold nanoparticle reagent on a sufficient scale for X-ray contrast imaging is more challenging because its synthesis in sufficient quantities is more complex, and results depend upon how effectively the targeting mechanism can deliver a visible dose to the target, we are working to develop this technology, and hope to incorporate it into future AuroVist products.

* Research use only. Not approved for clinical or human use.

References:

  • Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M, and Smilowitz, H. M.: Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol., 79, 248-253 (2006).

  • Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M., and Smilowitz, H. M.: In Vivo Vascular Casting. Microsc. Microanal., 11, (Suppl. 2: Proceedings); Price, R.; Kotula, P.; Marko, M.; Scott, J. H.; Vander Voort, G. F.; Nanilova, E.; Mah Lee Ng, M.; Smith, K.; Griffin, P.; Smith, P., and McKernan, S., Eds.; Cambridge University Press, New York, NY, p. 1216CD (2005).

  • Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M.: The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309-N315 (2004).

We are not the only ones interested in gold nanoparticles for medical imaging. A recent review by Sanvicens and Marco in Trends in Biotechnology highlights the growing use of nanoparticles in human health applications, including multifunctional nanoparticles, and highlights their advantages over conventional technologies.

Reference:

  • Sanvicens, N., and Marco, M. P.: Multifunctional nanoparticles - properties and prospects for their use in human medicine. Trends Biotechnol., 26, 425-433 (2008).

More information:

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S-Layer Proteins, Lipid Bilayers, and Nano-W

The list of publications citing our negative stains keeps growing and growing...this month, Keizer and co-workers use Nano-W to demonstrate the role of S-layer proteins in stabilizing lipid bilayer membranes, a property with potentially important nanobiotechnology implications. Nano-W (tungstate), and our other negative stain, NanoVan (vanadate) are non-reactive, amorphous, intermediate density stains, based on organic salts of tungsten and vanadium respectively, that are used to define the edges of particulate specimens for electron microscopic observation. Their highly amorphous structure and fine grain provides maximum clarity and least interference in the observation of ultrastructural features of interest. NanoVan is ideal for use with smaller gold labels such as Nanogold® because the stain is less electron-dense than other negative stains such as uranyl acetate or lead citrate, so sufficient contrast is produced between the gold particle, their environment, and the negative stain to differentiate them. They are particularly useful for studies of virus and protein ultrastructure.

Advantages of these reagents:

  • NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
  • Near-neutral pH results in better ultrastructural preservation.
  • NanoVan is less susceptible to electron beam damage than uranyl acetate.
  • Fine grain allows high imaging resolution.

[Negative Staining - Principle and Examples (41k)]

Schematic showing how negative stains work (left) and high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

S-layer proteins are commonly found in bacteria and archaea as two-dimensional, monomolecular crystalline arrays: they form the outermost cell membrane component, have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications where they could potentially be used to direct the assembly of other components. In this study, the authors show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer, and used them to isolate and study the activity of ion channels incorporated into S-layer protein-stabilized lipid bilayers.

Transmission electron microscopy (TEM) was used to visualize and compare the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membranes in the presence and absence of S-layers. Prior to re-crystallization, lyophilized B. sphaericus S-layer monomers were re-suspended in High Purity Water to an approximate concentration of 0.41 mg/mL. For re-crystallization from the 'bath side,' the S-layer protein, in ultrapure water, was added to a buffer solution (5 mM MOPS, 250 mM KCl and 0.1 mM CaCl2, pH 7.4) to obtain a final protein concentration of 0.2 mg/mL. 1mM CaCl2 solution was added to keep a [Ca2+] of 0.3 mM. A lipid/M2delta monolayer was formed by spreading a chloroform solution on the buffer solution surface. The pipette solution was diluted to the same concentration as the bath solution with ultra-pure water and 1mM CaCl2 solution. The bilayer membrane (BLM) was then formed using the tipdip methodology and the S-layer was crystallized on the bath side (Fig. 1). For the 'pipette side' configuration, the S-layer solution, was added to the micropipette together with the lipid and M2delta. The bilayer was then formed using tipdip methodology, and the S-layer protein self-assembled on the pipette side.

M2delta containing lipid vesicles were prepared by mixing a lipid solution (2 mg/mL in chloroform) containing DPhPC and DPhPE in a 7:3 molar ratio with M2delta (dissolved in methanol) for a final lipid to M2delta molar ratio of 1000 : 1. The solvents were evaporated under vacuum and the lipidprotein mixture hydrated in 1 mL water and heated at 50°C until a clear solution resulted (ca. 1 h). The suspension was allowed to cool to room temperature, then sonicated in a bath for 5 minutes and filtered through a 0.45-µm syringe filter. Vesicle size was determined by dynamic light scattering: typically, vesicle diameters of 137 ± 30 nm were found. The vesicles were added to the Teflon bath (0.8 cm in diameter) and mixed with Calcium-free buffer solution (5 mM MOPS, 250 mM KCl, pH 7.4 pH 7.4) and S-layer protein (0.2 mg/ml). A monolayer was formed by spreading a chloroform solution (1.5 µL of a 5mg/mL lipid DPhPC : DPhPE, 7 : 3 molar ratio) on the bath solution to allow vesicle fusion and the formation of a bilayer. 0.4 µL of a 1M CaCl2 solution was added after 1 hour to initiate S-layer self-assembly. Sixteen hours later, the bilayer was collected on a carbon coated copper TEM grid (300mesh, formvar removed). The specimen was stained with Nano-W and examined by transmission electron microscopy using a Hitachi H-7000 microscope at an accelerating voltage of 75 kV. Scion Image was used to assist recognition of the S-layer lattice structure.

Increased lifetimes and stability were found for S-layer protein stabilized bilayer lipid membranes formed by tipdipmethodology. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes, and activity was recorded for up to 20 hours or longer. The M2delta ion channel characteristics were compared for BLMs of the same composition with and without S-layers: the S-layers were found to be non-intrusive to the channel functionality and characteristics. The ability of S-layer proteins to stabilize BLMs, especially those that enhance the stability of BLMs beyond the use of tethers or polymer supports, and their non-intrusive character on ion channel activity make them potentially ideal for biosensor applications.

Reference:

  • Keizer H. M.; Andersson M.; Chase C.; Laratta W. P.; Proemsey J. B.; Tabb J.; Long J. R, and Duran R. S.: Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes. Colloids Surf. B Biointerfaces, 65, 178-185 (2008).

More information:

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Ask for our New Catalog, and See Us at Microscopy and Microanalysis 2008

Our new product catalog and applications guide is now available. This includes many new applications in nanotechnology, biosensing, labeling and detection chemistry, as well as new products such as EnzMet and GoldiBlot, and incorporates all the new products that we have introduced since the previous edition, including GoldEnhance, Ni-NTA-Nanogold, and Alexa Fluor® FluoroNanogold conjugates.

The last few years have seen an explosion of new applications for gold nanoparticles in nanotechnology applications, and Nanogold and related products have been featured in many exciting publications in these fields. Our new catalog includes the key references and shows how the key features of Nanogold, such as its specific reactivity, solubility, and site-specific conjugation chemistry, have helped enable these novel uses.

Those of you who use our products for immunogold labeling will want to attend the Roundtable panel discussion on immunogold labeling at Microscopy and Microanalysis 2008, to be held from 1:30 to 3:30 pm on Tuesday, August 5. Frank Macaluso (Albert Einstein College of Medicine Analytical Imaging Facility), Paul Webster (House Ear Institute, Hong Yi (Emory University EM Core, and Rick Powell of Nanoprobes will be discussing how to obtain the best results from immunogold labeling.

More information:

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Other Recent Publications

The Alivisatos group continued their pioneering contributions to the field of nanobiotechnology in a recent article in the Journal of the American Chemical Society. The authors used enzymatic ligation of discrete nanoparticle-DNA conjugates to create nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than by double-stranded DNA as in previous experiments. Conjugates were prepared by the coordination of thiol-modified oligonucleotides to citrate-stabilized 5 nm colloidal gold treated with bis-(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt to allow phosphine ligands to replace the citrate ligands. Ligation by T4 DNA ligase was verified by agarose gel and small-angle X-ray scattering. This capability was utilized in two ways. Firstly, a new class of multiparticle building blocks for nanoscale self-assembly was prepared. Secondly, an enzymatic amplification system was developed in which larger quantities of gold-nanoparticle assemblies were prepared from monoconjugates in the presence of a small amount of template, thus providing a means to amplify a population of discrete nanoparticle assemblies.

Reference:

  • Claridge, S. A.; Mastroianni, A. J.; Au, Y. B.; Liang, H. W.; Micheel, C. M.; Fréchet, J. M., and Alivisatos, A. P.: Enzymatic ligation creates discrete multinanoparticle building blocks for self-assembly. J. Amer. Chem. Soc., 130, 9598-9605. (2008).

Also in the Journal of the American Chemical Society, Wu, Steigerwald et al. investigate the mechanism of photoconversion of aqueous 8 nm silver nanocrystal seeds, prepared by sodium borohydride reduction of silver nitrate, into 70 nm single crystal plate nanoprisms upon irradiation with standard fluorescent tube light. Spectroscopic and light scattering experiments indicate that the process relies on the excitation of silver surface plasmons. The process requires dioxygen, and the transformation rate is first-order in seed concentration. Although citrate is necessary for the conversion, and is consumed during reaction, the transformation rate was found to be independent of citrate concentration. A mechanism was proposed based on these observations: oxidative etching of the seed is coupled with subsequent photoreduction of aqueous Ag+, and the reduced silver deposits onto a silver prism of specific size that has a cathodic photovoltage resulting from plasmon "hot hole" citrate photo-oxidation. This photovoltage mechanism also explains recent experimental results involving single and dual wavelength irradiation and the core/shell synthesis of silver layers on gold seeds.

Reference:

  • Wu, X.; Redmond, P. L.; Liu, H.; Chen, Y.; Steigerwald, M., and Brus, L.: Photovoltage mechanism for room light conversion of citrate stabilized silver nanocrystal seeds to large nanoprisms. J. Amer. Chem. Soc.,, 130, 9500-9506 (2008).

To further the theme of metal nanoparticles in biomedical imaging, Jarrett and co-workers report a novel development in the current issue of Bioconjugate Chemistry: 64Cu-labeled magnetic nanoparticles for multimodal imaging. Gautier Complementary imaging modalities provide more information than either method alone: the new particles are used in dual-mode imaging probes for combined magnetic resonance (MR) and positron emission tomography (PET) imaging. The positron-emitting copper isotope 64Cu was conjugated to the surface of larger nanoparticles via chelation with functionalized aza-macrocycle 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA). Amine-coated polystyrene beads (78 nm or 1 µm) were derivatized using an amine-reactive (isothiocyanate) form of DOTA; Anionic dextran sulfate-coated iron oxide (ADIO) nanoparticles (33 - 36 nm) were activated using a ring-opening reaction with periodate to generate surface aldehyde groups, and these were then reacted with an amine-activated DOTA. Incorporation of chelated 64Cu to nanoparticles under these conditions, which is routinely used to couple DOTA to macromolecules, was unexpectedly difficult, and required copper chelation before conjugation. Labeling yields of 24% for the amine polystyrene beads, and 21% radiolabeling yield for the anionic dextran sulfate iron oxide nanoparticles, were obtained. The resulting conjugates are targeted to vascular inflammation, and the 64Cu-magnetic particles can be used as dual-mode PET/MRI active probes. This chemistry may also be applied to attaching other chelated metals to other nanoparticle platforms.

Reference:

  • Jarrett, B. R.; Gustafsson, B.; Kukis, D. L., and Louie, A. Y.: Synthesis of 64Cu-labeled magnetic nanoparticles for multimodal imaging. Bioconjug. Chem., 19, 1496-1504 (2008).

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