N A N O P R O B E S E - N E W S
Vol. 10, No. 9 September 30, 2009
Updated: September 30, 2009
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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Although the our new EnzMetTM reagent provides excellent results for in situ hybridization, Nanogold® in situ hybridization still provides important advantages for some applications. When used with the more selective GoldEnhance gold enhancement procedure (GOLDFISH) rather than silver enhancement, it provides much more specific detection of targets in the presence of metallic substrates, which can develop signals with silver in the absence of gold. Ehrhardt and co-workers, in their recent paper in the Journal of Microscopy have now demonstrated that this method is also ideal for the genetic identification of microbes associated with specific geological surfaces by environmental scanning electron microscopy (ESEM).
Nanogold® in situ hybridization provides higher sensitivity and improved resolution compared with conventional chromogenic in situ hybridization using DAB or other enzyme chromogens. Diffusion of the deposited metal is negligible, and the black signal is readily differentiated from other stains.
Upper left: Schematic of silver or gold-enhanced Nanogold-based in situ hybridization detection. Lower left: DAB vs. GoldEnhanceTM-Nanogold®: Formalin-fixed serial paraffin sections of cervical carcinoma, in situ hybridized for HPV-16/18 using a biotinylated probe (bar = 10 µm). (a) DAB-peroxidase; (b) Nanogold®-Streptavidin with GoldEnhanceTM (courtesy of G. W. Hacker, Medical Research Coordination Center, University of Salzburg). Right: Enhancement of Nanogold® by GoldEnhanceTM: mechanism. Final particle size is controlled by enhancement time; particles may be enlarged to sizes between 3 nm (1-2 minutes) and 50 nm or larger (10 minutes and longer).
Electron microscopic imaging of geomicrobial specimens is problematic because they are often associated with metallic substrates and surfaces which can obscure detection of Nanogold-labeled cells imaged with backscattered electron microscopy. In addition, metallic surfaces can also give rise to non-specific precipitation of silver during the conventional silver enhancement process, leading to non-specific background signal. A new method is needed to identify microbes found geomicrobiological environments with a high concentration of metal substrates, such as hydrothermal vents and acid mine drainage. The authors have developed a new method which provides increased concentrations of Nanogold probes bound to rRNA targets within the cell, and makes individual hybridization events directly visible with secondary electron SEM imaging.
For in situ hybridization, samples were incubated with 100 µL of alkali solution (0.5 M NaOH, 1.5 M NaCl) for 8 minutes at 37°C to allow gold labeling reagents to enter the cell, and neutralized with 100 µL of acidic solution (0.5 M HCl, 1.5 M NaCl). In order to permeabilize the cells, samples were then incubated in lysozyme solution (0.5 mg/ml lysozyme in 100 mM TrisHCl pH 7.5, 50 mM EDTA) for 8 minutes at 37°C, then rinsed with sterile ultrapure water. Permeabilization was achieved by incubating with 0.1 mg/mL of proteinase K (in 100 mM TrisHCl, pH 7.5, with 5 mM EDTA) for 5 minutes at room temperature. After hybridization with the specific rRNA probe, the specimens were rinsed with deionized water, and cells were then blocked with 100 µL of PBS containing 0.1% gelatin and 0.1% Tween-20. Nanogold®-Streptavidin, diluted 1 : 400 in 1% bovine serum albumin in PBS, was then applied and allowed to react for 30 minutes at room temperature. The specimens were soaked in PBS containing 0.1% gelatin and 0.1% Tween-20 at room temperature for 10 minutes, then in filtered deionized water. Gold autometallography was performed by incubating in 100 µL of GoldEnhanceTM EM for 15 minutes at room temperature in the dark. The filter sections were then soaked in PBS containing 0.1% gelatin and 0.1% Tween-20 for 10 minutes, and finally in filtered deionized water for 10 minutes.
This modified Nanogold® in situ hybridization technique was tested first on separate liquid cultures of Escherichia coli strain +pMMB67HE and Pseudomonas aeruginosa PG201. Exponential phase cultures for both organisms were grown in Luria Bertani broth and harvested after 8 hours in a shaking incubator (30°C, 200 rpm). Cells were washed in PBS, fixed in 4% paraformaldehyde (4°C, 3 hours), and spotted onto glass slides. The E. coli and P. aeruginosa-sand cultures were hybridized with Bacteria-specific rRNA probes EUB338 and NON338 respectively before labeling with Nanogold-Streptavidin: this confirmed that the method provided successful labeling.
To test the effectiveness of this method for detecting microbes attached to mineral surfaces and in biofilm communities, a small aliquot (~10 µL) of exponential phase P. aeruginosa PG201 was spiked into Luria Bertani broth (10 mL), mixed with 50 g sterile Accusand (75150 µm) and incubated for 24 hours (30°C, 200 rpm). Following incubation, ~1 g of wet culture slurry was removed, fixed and spotted onto glass slides as before. In addition, the Nanogold in situ hybridization technique was then evaluated with a hyperthermophilic archaeal culture (Pyrococcus GB-D) grown in the presence of microcrystals of pyrite and elemental sulfur, to determine the specificity of hybridization reagents for cells versus metallic mineral surfaces. The culture broth, containing Pyrococcus cells, pyrite and sulfur, was fixed in 4% paraformaldehyde and spotted onto glass slides as described above. Archaeal specific primer ARC915 was used, followed by Nanogold in situ hybridization on these cultures.
All Nanogold-hybridized samples were imaged using an environmental scanning electron microscope (ESEM) with a field emission gun operating at either 5 or 15 KeV. Imaging was conducted in both wet mode (3.94.4 torr) with a gaseous secondary electron detector or in high vacuum with a secondary electron detector. All samples were air-dried at room temperature before imaging. Morphologic and textural differences between Nanogold-labeled cells and negative control cells were quantified by variety analyses performed on the ESEM image. Variety values were calculated in ArcView 3.2a by using a 6 by 6 pixel kernel because this matched the scale of brightness variations seen in hybridization clusters of Nanogold particles.
Hybridization experiments on pure cultures of E. coli revealed clusters of Nanogold particles (~70150 nm) arranged in the size and shape of bacterial cells: the size, morphology and electron density (determined from secondary electron image) of individual bright features were consistent with enhanced Nanogold particles. In wet mode SEM (ca.4 torr), cell envelopes are not visible. Nanogold hybridizations with exponential phase P. aeruginosa cells and EUB338 probe gave similar results. In order to improve contrast and resolution, P. aeruginosa cells were carbon coated and visualized under high vacuum with the ESEM. Surface textures and morphology of Nanogold clusters were visible, as was a faint cell envelope surrounding the hybridized gold particles. For hybridizations on PG201-sand cultures, Nanogold-labeled cells were less easily detected compared than with pure cultures; however, locations of rRNA-targeted Nanogold hybridization were clearly identifiable by morphologically uniform, small (<150 nm) clusters of individual Nanogold particles. This demonstrates that hybridization can successfully target multi-cell aggregates or biofilm-like substances.
Because gold is homogeneously distributed on all surfaces throughout the sample, back-scattered electron imaging and energy dispersive X-ray spectroscopy microanalysis alone would not have distinguished between rRNA-bound Nanogold and gold precipitates on mineral surfaces. However, by removing the cell exterior and then examining morphological, textural and variety value differences, hybridization events could be distinguished from Nanogold clusters and gold precipitation occurring non-specifically at mineral surfaces, and this provides a method which may be used to identify microbes even in highly metallic environments.
- Ehrhardt, C. J.; Haymon, R. M.; Sievert, S. M., and Holden, P. A.: An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy. J. Microsc., 236, 5-10 (2009).
More details of methods:
- Kenzaka, T.; Ishidoshiro, A.; Yamaguchi, N.; Tani, K, and Nasu, M.: rRNA sequence-based scanning electron microscopic detection of bacteria. Appl Environ Microbiol., 71, 5523-5531 (2005).
- Gérard, E.; Guyot, F.; Philippot, P, and López-García, P.: Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy. J. Microbiol. Methods., 63, 20-28 (2005).
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We have updated the product instructions for our FluoroNanogoldTM line of combined fluorescent and gold probes. The revised instructions update probe concentrations, buffers, and protocols to those found to give the best performance.
These unique immunoprobes may be used for a number of correlative microscopy methods:
FluoroNanogold probes contain both the 1.4 nm Nanogold®; label and a fluorescent label (currently Alexa FluorTM* 488 or 594, or fluorescein, are available; other fluorescent labels are planned). Both labels are covalently linked to Fab' fragments or to streptavidin to give a probe with the same high penetration and antigen access as our Nanogold-Fab' fragments. In addition, the 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.
Top: Structure of Alexa Fluor 594 FluoroNanogold-Fab' (left) and streptavidin conjugates (right), showing covalent attachment of Nanogold and Alexa Fluor 594 labels. above: 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).
Since we offer both Alexa Fluor* 488 and Alexa Fluor* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.
Because of their unique dual nature, obtaining the best results may require optimization. One of the challenges in using these probes is that the optimum use conditions (concentration and buffer) for the two labels may be slightly different, and some compromise may be required to find the conditions that give the best results with the FluoroNanogold combined probe. In addition, control of non-specific binding may need to be quite rigorous since both labels can interact non-specifically with biological components.
We have found that the following methods may help to reduce background:
- The most effective blocking agent we have tested is 5% nonfat dried milk. This was found to be particularly effective when mixed with and added to the specimen together with the FluoroNanogold conjugate. Cold-water fish gelatin has also been found to be helpful for gold probes in general.
- Adjusting camera exposure: manual control of exposure can also help in reducing apparent background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates. Since these are larger and more highly labeled, they give brighter fluorescence. If automatic exposure adjustment is allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Setting the camera exposure manually can be used to overcome this effect.
- For reducing the background in electron microscopy, sodium citrate buffer was found to be more effective than other buffers when used as a wash before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation.
Gold enhancement has been shown to provide very low backgrounds in in situ hybridization and blotting. Therefore, if background is a problem with silver enhancement, we recommend gold enhancement as an alternative.
- Background binding is often attributed to hydrophobic interactions (both the gold and fluorescent labels have some hydrophobicity), and therefore adding reagents that reduce hydrophobic interactions to the wash buffer may help remove non-specific binding. Examples include:
- 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring (Reference: Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986)).
- 0.1 % to 1 % detergent, such as Tween-20, or Triton X-100. 0.1% saponin may also be useful since its effects are reversible, so ultrastructural preservation may be improved if it is removed in later steps.
- 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.
- Powell, R. D., and Hainfeld, J. F.: Combined fluorescent and gold probes for microscopic and morphological investigations. In: Gold and Silver Staining: Techniques in Molecular Morphology; Hacker G. W., and Gu, J. (Eds): CRC Press, Boca Raton, FL, 2002, Ch. 9, pp. 107-118.
- Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.: Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech., 42, 2-12 (1998).
* Alexa Fluor is a registered trademark of Invitrogen - Molecular Probes
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If FluoroNanogoldTM is not suited for your experiment but you are using a recombinant protein and need to correlate fluorescence with electron microscopic localization, an alternative is to deliver the fluorescent label as a Green Fluorescent Protein (GFP) fusion protein, then detect the target protein using a Nanogold® conjugate targeted to either the target protein or to GFP. Schulz and co-workers used this approach in their microscopic analysis of the centrosomal and nuclear envelope localization of endogenous Sun1 protein in Dictyostelium during interphase and mitosis.
Double labeling procedure using GFP and Nanogold: (a) expression of GFP fusion protein at target of interest(green); (b) application of Nanogold-labeled antibody against target protein or against GFP; (c) Silver or gold enhancement enlarges Nanogold particles for ease of visualization.
Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms. It usually involves Sun family proteins, which are located at the inner nuclear envelope. The behavior of Dictyostelium Sun1 during mitosis, the dynamics of linkage between centrosomes and nuclei, and the exact localization of endogenous Sun1 protein were unknown prior to this work. In order to investigate this, with the goal of deriving a general mechanism for how the outer nuclear membrane proteins interact with Sun1 in centrosome/nucleus attachment, the authors used Dictyostelium discoideum amoebae as a model to study centrosomal attachment to the nucleus, beginning their investigation with a functional characterization of Sun1.
To clarify the localization of Sun1, the authors constructed a GFP vector that allowed expression of the entire sun1 coding sequence as an N-terminal GFP-Sun1 fusion protein. They also raised a polyclonal antiserum directed against the Sun domain, in order to assess the localization of the endogenous protein by light and electron microscopy. Immunofluorescence microscopy revealed that endogenous Sun1 was localized at the nuclear envelope, with a clear concentration at the pericentrosomal region in interphase cells, and this pericentrosomal concentration was preserved throughout mitosis. Closer analysis of deconvolved immunofluorescence images of mitotic cells in metaphase, anaphase and telophase revealed that during metaphase and anaphase, the Sun1-labeled zone of the nuclear envelope formed a bright collar around the spindle poles, which at this mitotic stage have entered the nuclear envelope. Particularly between anaphase and telophase, Sun1 was also concentrated along streaks at the nuclear surface that originated from the Sun1-positive zone in the pericentrosomal area: double staining with anti-tubulin antibodies demonstrated that these Sun1-positive streaks matched closely with microtubules nestling to the nuclear envelope. In interphase cells, Sun1 was also found in a fluorescent dot overlapping with the centrosome. This was labeled by anti-DdCP224 antibodies, suggesting that Sun1 may act as a centrosomal component. Therefore, microtubule-free, isolated centrosomes were investigated, and it was found that each isolated centrosome was associated with a Sun1-positive dot at its side, indicating that Sun1 may be a centrosomal component.
To clarify its association with the centrosome, the ultrastructural distribution of Sun1 was investigated by immunoelectron microscopy. For electron microscopic localization of Sun1, Dictyostelium nuclei with attached centrosomes were isolated by lysis followed by filtration through 5 µm polycarbonate filters, and the cell extract was loaded onto a modified step gradient comprising 1 mL of 50% sucrose in gradient buffer (100 mM Na-PIPES, pH 6.9, 2 mM MgCl2, 2 mM dithiothreitol (DTT), and protease inhibitor cocktail, and 2 mL of 30% sucrose in gradient buffer in 15-mL tubes. The nuclei were sedimented at 3700 x g for 10 minutes at 4°C in a swing-out rotor. Nuclei at the interface between the 50% and 30% fraction were then collected with a pasteur pipette. For each specimen, approximately 2107 with GFP nuclei were suspended in 1 mL of 10 mM PIPES with MgCl2 and protease inhibitor cocktail, and sedimented onto 12-mm coverslips in 24-well plates by centrifugation (2800 x g for 10 minutes at 4°C). Nuclei were fixed with 0.5% glutaraldehyde and treated with 0.1% sodium borohydrate in phosphate-buffered saline (PBS). Samples were blocked with 0.5% bovine serum albumin (BSA) in PBS, then incubated with anti-Sun1 or preimmune antibodies (1 : 500 dilution), followed by incubation for 1 hour with Nanogold-Fab? anti-rabbit IgG, diluted 1 : 25 in blocking solution. After Nanogold labeling, samples were washed three times for 5 minutes in PBS, postfixed for 15 minutes with 2% glutaraldehyde in PBS, then washed again in PBS and water. Signals were intensified for 45 minutes at room temperature using a commercial silver enhancement kit (R-Gent SE-EM, Aurion). After further washing (4 x 5 minutes) in water and gold-toning, specimens were osmicated for 15 minutes in 0.5% OsO4 in 0.1 M phosphate buffer (pH 7.0), rinsed in water, dehydrated in a graded series of ethanol, and embedded in Spurr's resin. Sections were stained with uranyl acetate (5 minutes) and lead citrate (2 minutes), then examined in a Philips CM100 electron microscope.
As expected, gold particles were distributed mainly in the pericentrosomal quadrant of the nuclear envelope, close to both nuclear membranes, with even a slight bias toward the outer nuclear membrane, but specific staining was not found at the centrosome itself. Therefore, the positive staining was ascribed to associated patches of outer nuclear membrane.
Disruption of Sun1 function by overexpression of full-length GFP-Sun1 or a GFP-Sun-domain deletion construct revealed not only the established function in centrosome/nucleus attachment and maintenance of ploidy, but also that Sun1 is required for the association of the centromere cluster with the centrosome. Live-cell imaging was used to visualize the occurrence of mitotic defects: this showed that microtubules are required for dynamic distance changes between centrosomes and nuclei. FRAP analysis then revealed at least two populations of Sun1: an immobile fraction associated with the centrosome, and a mobile fraction in the nuclear envelope. The authors propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium.
- Schulz, I.; Baumann, O.; Samereier, M.; Zoglmeier, C, and Gräf, R.: Dictyostelium Sun1 is a dynamic membrane protein of both nuclear membranes and required for centrosomal association with clustered centromeres. Eur. J. Cell. Biol., 88, 621-638 (2009).
Gold toning method:
- Laube, G., Roper,J., Pitt, J. C., Sewing, S., Kistner, U., Garner, C. C., Pongs, O., Veh, R. W.: Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux. Brain Res. Mol. Brain Res., 42, 5161 (1996).
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AuroVistTM is the first gold nanoparticle-based X-ray contrast agent. It is a 1.9 nm stabilized gold particle that provides up to ten times the contrast of iodine-based reagents for both micro-CT and clinical CT applications.* At appropriate beam energies (typically just above the gold L and K edges), gold achieves a contrast up to three times greater than iodine per unit mass, yielding initial blood contrast greater than 500 Hounsfeld Units (HU). It may also be injected in concentrations up to four times those of iodine: combining these factors yields a total contrast gain of up to ten times or more.
With AuroVistTM, You can obtain high-resolution, high-contrast images of blood vessels, organs, other anatomical structures and tumors in animals. AuroVistTM is highly soluble, biocompatible, and stable to the environment found in the vascular system and in tissues. Unlike many iodine reagents, it also has very low viscosity and osmolality. This means less traumatic injections, and the ability to inject into smaller blood vessels with much lower risk of damage. AuroVistTM gives you these enhanced performance features:
- 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).
- Clears through kidneys. Kidney fine structure may be imaged from 1-2 minutes up to an hour or more after injection; concentration in the kidneys can provide contrast values as high as 1,500 HU or more.
- 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.
(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).
* Research use only. Not approved for clinical or human use.
AuroVistTM 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 with this reagent.
How much should I use?
With AuroVistTM, the amount you need and the number of applications which may be performed with one vial depends upon the degree of contrast you require and the properties of the feature you wish to image (size, location in the body, depth), and also the time taken for the AuroVist to reach the target: are you imaging vascular features close to the administration site, in which delivery is immediate, or organs in which the reagent accumulates or passes through, such as the kidneys?
In their original paper, Hainfeld and co-workers used a high concentration of AuroVistTM, and it is probably more reasonable to start with a lower concentration. In addition, we have found that different strains of mice may show different LD50 values, and for some outbred strains, the LD50 may be lower than the Balb/C, nude and C3H strains used in our experiments.
One vial of AuroVistTM contains 40 mg of gold. If you are conducting vascular imaging, we recommend that you use a concentration that keeps the total amount injected below about 1.4 g Au/kg body weight. In our experiments, some strains of outbred mice have shown LD50 values close to this. The average mouse is about 20 g ; therefore, to achieve a body concentration of 1.4 g Au/kg body weight, you should inject 28 mg or less. If you were to use 20 mg, you would obtain two doses from one vial.
Significantly lower doses may yield useful contrast. For example, we have obtained usable images of mouse kidneys using microCT at concentrations as low as 30 mg Au/kg - 100 times less than the LD50. Therefore, a good compromise for many organs may be to start with a value that is about one-fifth of the cited LD50 value, or about 280 mg Au/kg; this would require 5.6 mg of gold, and allow up to 7 doses per vial.
What instrument and beam settings should I use?
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.
This must be compared this with the absorption spectrum of soft tissue. X-ray absorption spectra for elements and tissues are available from NIST:
X-ray absorption spectra
- Mammography: These instruments are suitable for small animal imaging. Use of lower kVp, at or below the L-edge absorptions of gold at ~13 keV, or ~40 kVp (e.g., 22 kVp) is recommended to take advantage of the L edge gold 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 (typically 120 kVp or higher). 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. To enhance detection, or give the best absorptive contrast, the ideal settings are those that overlap the L-edge absorptions of gold at ~13 keV; this requires a kVp of about 3 times this or ~40 kVp - so the first choice for kVp setting would be 40 kVp, or as close as the instrument allows.
How can I ensure minimal toxicity?
Certain strains of mice appear to be more tolerant of this gold. 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:
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.
- Start with a moderate dose, such as 120 or 160 mg/mL (for 0.2 mL of solution). 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, and a modest reduction in dose can significantly reduce toxicity without compromising your results.
The average mouse has a mass of about 20 g, and a circulatory system volume of about 1 mL. The amount of AuroVist necessary to achieve some of the suggested g Au/kg values is as shown below:
|Mouse body mass
||Gold dose, g Au/kg
||Amount of AuroVist required, mg
||Injection volume at 200 mg Au/mL
||28 µL (0.028 mL)
||70 µL (0.07 mL)
||140 µL (0.14 mL)
||200 µL (0.2 mL)
||320 µL (0.32 mL)*
* Use more concentrated solution: keep injection volume to 0.2 mL or less.
- 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 have received several inquiries about whether a targeted version of this reagent is available. The preparation of a targeted gold nanoparticle reagent on a sufficient scale for X-ray contrast imaging is challenging both because of the greater complexity of the synthesis, and also because of the amount of reagent required for visualization. This depends on the size and target density of the feature to be imaged, and also on how effectively the targeting mechanism can deliver a visible dose to the target, and this may be less than preliminary studies such as blots or cell studies suggest. Other studies described in the literature indicate that the targeting methods investigated so far direct at most 15 - 25% of the reagent to the target site. However, we are working to develop this technology, and hope to incorporate it into future AuroVistTM products.
- 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).
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Nanoprobes has received another new Small Business Innovation (SBIR) Research grant, this time from the National Institute of Mental Health (one of the National Institutes of Health (NIH)), to develop electron microscopic and labeling methods for studying nervous system plasticity and the connectivity of the nervous system.
Our objective is to develop technology for the study of neural circuits in depth, which will provide insight into the normal wiring of the nervous system as well as into how this wiring can change or go awry. This in turn will provide fundamental insights into the processes that underlie many disorders of the nervous system suspected to be due to defects in the connections between neurons. These include autism, Down syndrome, fragile X syndrome, Alzheimer's disease, epilepsy, Guillain-Barre syndrome, multiple sclerosis (which affects more than 400,000 Americans), muscular dystrophy, and Parkinson's disease (which is estimated to affect 1 million people in the USA and 6 million worldwide).
The 2-year Phase 1 study will provide funding for the development of immunogold reagents for use in serial blockface scanning electron microscopy (SEM), a novel method which is expected to provide high-resolution localization of components of small features such as gap junctions in large volume specimens of nervous tissue. The work will be directed by Dr. Richard Powell, and conducted with Dr. Vishwas Joshi in collaboration with Dr. Eduardo Rosa-Molinar of the Biological Imaging Group at the University of Puerto Rico, Rio Piedras.
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If you have been unable to attempt cryofixation because you don't have the equipment, Leunissen and Yi may have the answer in a recent Journal of Microscopy paper - self-pressurized rapid freezing (SPRF), a novel cryofixation method for electron microscopy that provides a drastic simplification. In SPRF, specimens are sealed in a capillary tube, then plunge frozen in a cryogen such as liquid propane or liquid nitrogen. A number of single-cell test specimens were found to be well preserved using this method, and multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The authors found the preservation of these unprotected specimens to be comparable to that achieved using high-pressure freezing in the presence of cryoprotectant. The excellent cryopreservation appears to arise from pressure build-up which occurs upon the cooling of water below its melting point in a confined space, possibly from the conversion of a fraction of the water content into low-density hexagonal ice, or expansion of water during supercooling. Calculations indicate the pressure may be similar to that applied in high-pressure freezing. A range of cryogens and cryogen temperatures were used successfully. Because the pressure is generated inside the specimen holders by cooling rather than applied from an external source, the technique has been dubbed self-pressurized rapid freezing.
We have previously described the use of gold nanoparticles for enhancement of radiotherapy, and in a recent Journal of Controlled Release paper, Jeong and group provide a slightly different take on using gold particles for cancer therapy: as carriers for delivery of a radiosensitizer to cancer cells. Effective delivery of a radiosensitizer to target tumor cells, which preferentially increases tumor cytotoxicity while simultaneously minimizing damage to healthy cells around the tumor, is an ideal strategy for the improvement of radiotherapeutic efficacy. The authors pursued enhanced radiotherapeutic efficacy using biocompatible gold nanoparticles (AuNP) as a vehicle for the systemic delivery of beta-lapachone (lap; 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione)), a novel anticancer drug that was originally isolated from the bark of the Lapacho tree. Lap is a novel anticancer agent that demonstrates potent cytotoxicity against cancer cells expressing NAD(P)H:quinone oxidoreductase-1 enzyme (NQO1), but a critical obstacle to clinical application of lap is its poor solubility and non-specific distribution.
- Leunissen, J. L. M., and Yi, H.: Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy. J. Microsc., 235, 25-35 (2009).
To avoid this problem, the authors evaluated gold nanoparticles carrying lap (AuNPs/lap) for active targeting of tumor cells and improving in vivo radiotherapeutic efficacy. AuNPs composed of poly(ethylene glycol) (PEG) shell, per-6-thio-beta-cyclodextrin (SH-CD) as a drug pocket, and anti-epidermal growth factor receptor (EGFR) antibody as a targeting ligand were prepared. AuNP carriers functionalized with CD and PEG (AuNP-1) were prepared by stirring a citrate-stabilized aqueous solution of AuNPs (average diameter ~27 nm) for 12 hours with SH-CD and a-methoxy-omega-mercapto-poly(ethylene glycol) (mPEG-SH, MW 2000 Da). Intermediate AuNP, which contains succinimidyl-functionalized PEG and CD, was prepared by the reaction of AuNP-0 with SH-CD, mPEG-SH, and alpha-succinimidyl propionate-omega-lipoic acid-poly(ethylene glycol) (NHS-PEG-SH). Intermediate AuNP was incubated with murine monoclonal anti-EGFR antibody (F4) in HEPES, pH 7.4 for 12 hours. The mixture containing AuNP-2 was then purified by ultracentrifugation and washing with water and the amount of anti-EGFR determined by western blot analysis. To load lap onto the surface, an excess amount of lap was added to Au nanocarrier solutions and sonicated for 3 minutes. AuNP-1/lap and AuNP-2/lap were purified by filtration and ultracentrifugation respectively. The AuNPs/lap was adjusted to 4 mg/mL (w/v) in lap. The average hydrodynamic diameters of AuNP-0, AuNP-1, AuNP-2, AuNP-1/lap, and AuNP-2/lap were measured by dynamic light scattering (DLS) to be 28.9 nm, 40.4 nm, 46.7 nm, 42.6 nm, and 47.0 nm, respectively. Murine monoclonal anti-EGFR antibody was conjugated to the AuNPs/lap in order to target the nanoparticles: the active tumor-targeting property of AuNPs/lap conjugated with anti-EGFR antibody was validated in in vitro experiments, using cell lines expressing EGFR at different levels. In mice bearing xenograft human tumors, the intravenous injection of AuNPs/lap greatly enhanced radiotherapeutic efficacy. The AuNP/lap approach therefore offers a new modality for improvement of radiotherapeutic efficacy, and may make feasible the clinical application of lap for human cancer treatment.
- Jeong, S. Y.; Park, S. J.; Yoon, S. M.; Jung, J.; Woo, H. N.; Yi, S. L.; Song, S. Y.; Park, H. J.; Kim, C.; Lee, J. S.; Lee, J. S., and Choi, E. K.: Systemic delivery and preclinical evaluation of Au nanoparticle containing beta-lapachone for radiosensitization. J. control. Release, 139, 239-245 (2009).
The localization and microspectroscopic characterization of metallic species in cells is reviewed by Ortega and colleagues in the Journal of the Royal Society - Interface. The direct detection of biologically relevant metals in single cells and of their speciation is a challenging task that requires sophisticated analytical methodology. The article presents recent achievements in the field of cellular chemical element imaging and direct speciation analysis, using proton and synchrotron radiation X-ray micro- and nanoanalysis. Recent improvements in focusing optics for MeV-accelerated particles and keV X-rays allow the application of these methods to chemical element analysis in subcellular compartments. In addition, the imaging and quantification of trace elements in single cells using particle-induced X-ray emission (PIXE) is reviewed: in combination with backscattering spectrometry and scanning transmission ion microscopy, PIXE provides high accuracy in elemental quantification of cellular organelles. By comparison, synchrotron radiation X-ray fluorescence provides chemical element imaging with less than 100 nm spatial resolution, but also offers the unique capability of spatially resolved chemical speciation using micro-X-ray absorption spectroscopy. Examples of the potential applications of these methods in biomedical investigations include cellular toxicology, pharmacology, bio-metal analysis and mapping the distribution of metal-based nanoparticles.
- Ortega, R.; Devès, G., and Carmona, A.: Bio-metals imaging and speciation in cells using proton and synchrotron radiation X-ray microspectroscopy J. R. Soc. Interface, 6, S649-S658 (2009).
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