N A N O P R O B E S     E - N E W S

Vol. 8, No. 3          March 13, 2007

AuroVist 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 you these enhanced performance features:

For microCT, beam energy should be just above golds 12-14-keV L-edge, while for clinical CT it can be tuned to just above gold's 80.7-keV K-edge. Unlike iodine, AuroVist has very low viscosity and osmolality, and therefore may be injected and used in small blood vessels without risk of vascular damage.

Using this approach with a prototype of AuroVist, 20 µm blood vessels in live animals have been successfully imaged with microCT, and vascular casts obtained for the first time. Small orthotopic colon tumors were also detected in vivo using gold nanoparticles and microCT. Kidney fine structure may be imaged up to an hour or more after injection; concentration in the kidneys can provide contrast values as high as 1,500 HU or more. AuroVist has demonstrated low acute toxicities, and phamacokinetics are acceptable.

* 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).
  • Abstract (courtesy of the British Journal of Radiology).

• 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).
  • Paper

• 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).
  • Abstract (courtesy of Physics in Medicine and Biology).

More information:

• AuroVist: Catalog information
• AuroVist: Product information, instructions and protocols
• Technical help: AuroVist
• AuroVist: References sorted by application
• In Vivo Vascular Casting: our paper from Microscopy and Microanlaysis 2005

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Using AuroVist: Tips for Success

AuroVist is supplied as a dry solid. It is highly soluble in water and aqueous buffers. To use, add water, PBS, or other desired buffer to the dried gold nanoparticles. They should dissolve rapidly. Next, filter through a 0.2 micron filter; a centrifugal filter is supplied that has less loss than syringe filters. Spin at 15,000 x g for 8 min. To ensure maximum recovery, a second and third filtration should be performed by adding 10-20 microliters of buffer or water to the filter and re-filtering to wash the membrane. A typical amount for intravenous injection, for example into the tail vein of a mouse, is 0.2 mL. Since the LD50 is > 1.4 g Au/kg, a 20 g mouse could be injected with 28 mg Au. For 28 mg in 0.2 ml, one would therefore dissolve the gold nanoparticles (vial contains 40 mg Au) in 0.36 mL of buffer and inject 0.2 mL.

In our development of this reagent, the following considerations were found to be significant.

What are the best instrument and beam settings to 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, and the settings below are appropriate for the different instruments:

• 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 down such that only 1 revolution is done in the selected imaging time (e.g., 1 hour). If the animal moves during collection of this data set, the back projection 3D reconstruction will be errant. This places significant constraints for live animal imaging, and motion must be minimized, such as breathing and heartbeat (mouse = 600 beats/min). A simple solution is to kill the animal some time after injection and then image, 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.

Toxicity is worse than I expected

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:

• 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, and a modest reduction in dose 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.

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).
  • Abstract (courtesy of the British Journal of Radiology).

• 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).
  • Paper

More information:

• AuroVist: Product information, instructions and protocols
• Technical help: AuroVist
• AuroVist: References sorted by application
• In Vivo Vascular Casting: our paper from Microscopy and Microanalysis 2005

What does Retinoschisin do? Find Out with Nanogold®

To determine the cellular distribution of retinoschisin, subcellular fractions and fractions enriched in photoreceptor inner and outer segments were prepared from mouse retina by differential or density gradient ultracentrifugation. Immunoblot analysis was used to assess the expression of RS in various subcellular compartments and its fractionation into soluble phase on treatment of retinal cell membranes with different solubilizing reagents. RSlipid interactions were evaluated by a proteinlipid overlay assay that used wild-type and mutant forms of RS discoidin domain glutathione S-transferase (GST) fusion proteins.

The subcellular localization of RS in mouse retina was visualized by pre-embedding immunogold electron microscopy, and the ultrastructure was evaluated by transmission electron microscopy. For electron microscopy, mice were deeply anesthetized with ketamine (50 mg/kg) and xylazine (5mg/kg) and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Eyes were enucleated and hemisected. The posterior eye cup segments were immersed in fixative for 2 hours and rinsed in phosphate-buffered saline (PBS) at 4°C overnight. For transmission electron microscopy, the fixed eye cups were dehydrated in ethanol in series (30%, 50%, 70%, and 96%), block-stained in 1% uranyl acetate in absolute ethanol for 1 hour, then rinsed twice in absolute ethanol and embedded via propylene oxide in epoxy resin (Embed 812). Ultrathin sections were cut and poststained in uranyl acetate and lead citrate. For immunoelectron microscopy, wild-type eye cups were embedded in 5% agarose/PBS and sectioned at 50-µm thickness on a microtome, and then processed for pre-embedding immunoelectron microscopy. Microtome-cut sections were preincubated in 10% normal goat serum (NGS) diluted in PBS for 2 hours, then incubated for 48 hours at 4°C in an anti-RS antibody diluted in PBS with 1% NGS. Next, the sections were incubated in a mixture of goat Nanogold-Fab' anti-rabbit affinity-purified Fab' fragments (1: 100). Gold particles were enhanced by silver amplification for 8 to 12 minutes with HQ Silver. Sections were treated with 1% OsO₄ and contrasted in 1% uranyl acetate before embedding. Serial electron microscopic sections were cut and collected on polyvinyl formal-coated copper slot grids and observed in the electron microscope.

RS was found to be intimately associated with cell membranes of the retina, and was shown to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, characteristic of a peripheral membrane protein, and was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Proteinlipid overlay assays indicated a preferential interaction between RS and anionic phospholipids. Extraction of RS from the membrane was found to be inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS^-/y mice, which failed to express RS protein.

Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane, through anionic phospholipids and divalent cations. This localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.

Reference:

• Vijayasarathy. C.; Takada. Y.; Zeng. Y.; Bush. R. A., and Sieving, P. A.: Retinoschisin is a peripheral membrane protein with affinity for anionic phospholipids and affected by divalent cations. Invest. Ophthalmol. Vis. Sci., 48 991-1000 (2007).
  • Abstract (courtesy of Investigative Ophthalmology and Visual Science).

More information:

• Catalog information: Nanogold conjugates
• Nanogold-IgG and Fab' conjugates: Product information and instructions
• Nanogold conjugates: technical help
• Nanogold references, sorted by application
• Silver enhancement: catalog information
• HQ Silver: Product information and instructions
• Silver enhancement: technical help

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Nano-W Helps Screen Inhibitors of Amyloid Beta Toxicity

The first method takes advantage of the unique ability of extracellularly applied ABeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). ABeta42 oligomers were found to induce a significant, concentration-dependent increase in MTT-MTT formazan exocytosis (MTT-FE) in up to 100% of cells. Unaggregated and fibrillar ABeta induced much smaller degrees of MTT. The small degree of MTT-FE induced by unaggregated ABeta at higher concentrations was possibly related to ABeta oligomers formed during incubation, which could be demonstrated by size exclusion chromatography and electron microscopy. Intracellular MTT forms granules which are effectively dissolved by 1% Tween-20; however, exocytosed MTT forms long, needle-shaped crystals on the cell surface, which are unaffected by Tween-20 but dissolve readily in isopropanol. This behavior provides two means to assay the degree of exocytosis of MTT: by visual analysis of the crystals, or by the ratio of MTT in the two fractions obtained by sequential Tween-20 and isopropanol extractions.

In the standard 2 hour assay protocol for compound testing, Neuro-2a (N2a) cells were plated at a density of 20,000 cells/well in 96-well plates in 100 µL of Opti-MEM. After overnight incubation, ABeta42 oligomers and compounds of specified amounts were added to the cultures. After incubation for 1 hour at 37°C, MTT (0.5 mg/ml) was added and the cultures incubated for a further 1 hour. At the conclusion of the assay, the cultures were photographed. The exocytosed MTT presented as needle-like crystals on the cell surface, clearly distinguishable from intracellular MTT granules. The percentage of cells exocytosing MTT formazan was determined by counting 300 cells in multiple fields as described. For sequential solubilization of MTT formazan, intracellular MTT granules were first solubilized by 1% Tween-20 at 37°C for 10 minutes with shaking. Solubilized MTT formazan in the supernatant was transferred to a new plate as the Tween-20-soluble MTT (TS-MTT). The remaining cell surface needlelike crystals were solubilized with 100% Isopropanol as Tween 20-insoluble MTT (TI-MTT). Absorbance values at 590 nm were determined for each fraction using 630 nm as the reference wavelength.

Samples of Abeta oligomer solutions were divided for AFM and EM studies. Particle size and number were determined using AFM as described previously. The sample was diluted into 50 mM Tris-HCl, pH 7 and spotted onto freshly cleaved mica. After two minutes the sample was washed with freshly distilled water, partially dried with compressed air, then dried completely at room temperature. Samples were imaged in air with a digital multimode Nanoscope III scanning probe microscope operated in tapping mode, and the grains analyzed using the SPIP imaging program to calculate the total number of fibrillar and non-fibrillar (spherical, annular and amorphous) assemblies in the 3 µm² AFM images. For each experimental condition, representative 3 µm² images obtained in three independent experiments were used to determine the average and standard deviation for particle density (number of particles per field) of fibrils and of spherical, annular and amorphous structures at 1 and 5 hours. In order to determine whether the increase in fibril density from one to five hours was significant, a Student's t-test was performed: representative 3 µm² AFM images were analyzed with SPIP to determine the range and average length, width and height of the spherical, annular and fibrillar structures observed under selected experimental conditions.

To confirm the results, negative stain transmission electron microscopy (TEM) was performed on samples of 3 µL, applied to a charged grid and allowed to settle for 10 seconds. The solution was then removed by blotting with a piece of filter paper and the sample washed once with 3 µl of water, then stained with a mixture of Nano-W (methylamine tungstate) and 1% trehalose. After 2 seconds, the staining solution was blotted off with a piece of filter paper. The staining procedure was repeated 3 times; tobacco mosaic virus (TMV) was then added and used as an internal control and for calibration, and the samples were observed in the electron microscope.

two novel inhibitors, code-named CP2 and A5, were quickly identified by the MTT-FE assay from two compound libraries. A second independent screen of the same libraries using a previously published MC65 protection assay, which identifies inhibitors of toxicity based on their ability to protect MC65 cells from ABeta and cell death, also selected the same two leads; this suggests that both assays select for the same anti-A? oligomer properties displayed by these compounds, and imply that they may be useful as a screening test for inhibitors of ABeta toxicity.

Reference:

• Hong, H. S.; Maezawa, I.; Yao, N.; Xu, B.; Diaz-Avalos, R.; Rana, S.; Hua, D. H.; Cheng, R. H.; Lam, KS., and Jin, L. W.: Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity. Brain Res., 1130, 223-234 (2007).
  • Abstract (courtesy of Science Direct).

Reference for MC65 protection assay:

• Maezawa, I.; Hong, H.-S.; Wu, H.-C.; Battina, S. K.; Rana, S.; Iwamoto, T.; Radke, G. A., Pettersson, E.; Martin, G. M.; Hua, D. H., and Jin, L. W. A novel tricyclic pyrone compound ameliorates cell death associated with intracellular amyloid-Beta oligomeric complexes. J. Neurochem., 98, 5767 (2006).
  • Abstract (courtesy of Journal of Neurochemistry).

Nanoprobes offers two novel negative staining reagents, NanoVan, which is based on vanadium, and Nano-W, which is based on tungsten. Because of the difference in the atomic numbers and hence staining density of these two elements, these two stains can be mixed to enable negative staining with a range of different densities. NanoVan is recommended for use with Nanogold® because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate, and does not obscure the Nanogold particles. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels.

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, amorphous staining allows high imaging resolution.

More information:

• Catalog information: Negative stain reagents
• NanoVan Negative stain: Our paper from Microscopy and Microanalysis 1994
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• Technical help: Negative staining
• Negative staining: References

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Nanoprobes at First Australian Correlative Microscopy Workshop

The workshop will focus on applications and advances in correlative microscopy techniques in biological sciences. Talks and practical demonstrations will cover different microscopies, such as optical, confocal, transmission electron microscopy, scanning electron microscopy and electron tomography, as well as providing information about sample preparation methods for correlative biomolecular imaging at the cellular and molecular level. Practical tips will be included on how to find the same area of interest in your sample in different microscopes, as well as how to enhance the size of gold particles for visualization by different methods.

The workshop will be held on Wednesday, 4 April 2007 from 09:15 to 17:00 at the Electron Microscope Unit, LG 92, Madsen Building F09 at The University of Sydney. Registration deadline is March 28. To register, contact Dr. Deborah Barton (telephone +61 2 9351 5220, fax +61 2 9351 7682; e-mail debbie.barton¤emu.usyd.edu.au).

More information:

• FluoroNanogold: introduction and catalog information
• FluoroNanogold: technical help
• Nanoprobes: Other News
• About us

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Other Recent Publications

Reference:

• Stone, J. W.; Sisco, P. N.; Goldsmith, E. C.; Baxter, S. C., and Murphy, C. J.: Using gold nanorods to probe cell-induced collagen deformation. Nano Lett., 7, 116-119 (2007).
  • Abstract (courtesy of ACS Publications).

Reference:

• Montalti M.; Zaccheroni N.; Prodi L.; O'reilly N., and James, S. L.: Enhanced Sensitized NIR Luminescence from Gold Nanoparticles via Energy Transfer from Surface-Bound Fluorophores. J. Amer. Chem. Soc., 129, 2418-2419 (2007).
  • Abstract (courtesy of ACS Publications).

A few weeks earlier, Aslan, Lakowicz and group described another advance in the development of metal enhanced fluorescence (MEF) and single nanoparticle sensing platforms, using core-shell silica-coated silver nanoparticles. Silver nanoparticles 130 ± 10 nm in diameter were prepared by sodium citrate reduction of silver nitrate. The silica shell was varied in thickness of the silica shell from 2 to 35 ± 1 nm, to optimize fluorescence enhancement; this was controlled by the concentration of tetraethoxysilane (TEOS) after alkaline initiation. By conjugating rhodamine 800, then etching the silver core with cyanide, the fluorescence in the presence and absence of the silver cores could be compared: enhancements of 8-fold to 20-fold were found with the silver cores present. Significant lifetime reductions were also found, and combining the faster cycling that is thus enabled with the fluorescence enhancements may provide up to a 200-fold increase in overall detectability. Fluorophores could be either covalently conjugated, or noncovalently doped into the silica layer. This approach was shown to be highly versatile and potentially important for single nanoparticle sensing applications.

Reference:

• Aslan, K.; Wu, M.; Lakowicz, J. R., and Geddes, C. D.: Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms. J. Amer. Chem. Soc., 129, 1524-1525 (2007).
  • Abstract (courtesy of ACS Publications).

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