Updated: March 24, 2000

NANOGOLD® PRODUCT INFORMATION

Nanogold®, Gold Particles, Non-Functional (NG)[Nanogold®, Gold Particles, Non-Functional (NG) product page]Product Name: NANOGOLD® (NG)Catalog Numbers:    2010Appearance:Brown solidRevision:1.3 (March 2000)

Technical Assistance Online  Instructions (PDF)  Material Safety Data Sheet (PDF)

General InformationNANOGOLD® (NG) is a revolutionary new type of gold particle, prepared using a process that gives precise control over its size distribution and surface properties. NANOGOLD® particles are a uniform 1.4 nm in diameter, making them a suitable calibration standard for electron microscopy. They do not aggregate, as do colloidal gold products, nor do they possess affinity for proteins as colloidal gold particles do.¹ This product does not posess active functionality: it cannot be linked specifically to proteins or antibodies. It is anticipated that this product will be used as a size or resolution standard for the electron microscope.

NANOGOLD® particles should be frozen upon receipt, and stored at -20°C.

Warning:For research use only. Not recommended or intended for diagnosis of disease in humans or animals.Do not use internally or externally in humans or animals.Non radioactive and non carcinogenic.Product SpecificationsNANOGOLD® is supplied as a solid, lyophilized from methanol solution. It is purified by gel filtration, and is stable under a wide range of pH conditions. It is soluble in alcohols, acetone, dichlorometane and similar solvents; it is also soluble in aqueous buffer systems such as phosphate buffered saline (150 mM NaCl).Extinction coefficients at specific wavelengths are given below for a methanol solution:

WAVELENGTH (nm)EXTINCTION COEFFICIENT*2802.25 X 10⁵4201.12 X 10⁵*Measured for 5 X 10^-6 M solution in methanol. Using Stains with Nanogold®Because the 1.4 nm NANOGOLD® particles are so small, over staining with OsO₄, uranyl acetate or lead citrate may tend to obscure direct visualization of individual NANOGOLD® particles.Three recommendations for improved visibility of NANOGOLD® are:

1. Use of reduced amounts or concentrations of usual stains.
2. Use of lower atomic number stains such as NANOVAN, a Vanadium based stain.²
3. Enhancement of NANOGOLD® with silver developers, such as LI SILVER or HQ SILVER.

ContentsTemperature CautionAlthough NANOGOLD® is usually stable,³ under some conditions labeled specimens or conjugates may not be stable above 50°C. Best results are obtained at room temperature or 4°C.Avoid 37°C incubations.Use low temperature embedding media (e.g., Lowicryl) if labeling before embedding;⁴ do not bake tissue blocks with NANOGOLD®. If your experiment requires higher temperature embedding, then silver enhancement should be performed before embedding.Thiol CautionCAUTION: NANOGOLD® particles degrade upon exposure to concentrated thiols such as beta-mercaptoethanol or dithiothreitol. If such reagents must be used, concentrations should be kept below 1 mM and exposure restricted to 10 minutes or less. Instructions for UseThe product is supplied as 10 nmol of solid. If you require less than this, then dissolve the sample in 1.0 mL methanol and pipette the required amount into a polyethylene tube. The methanol may then be blown off using nitrogen; the dark brown residue may be dissolved in the required solvent. A methanol solution of NANOGOLD® particles is stable for several months at 2 - 8°C. Special Considerations for Direct Viewing of Nanogold® in the Electron MicroscopeFor most work, silver enhancement is recommended to give a good signal in the electron microscope (see below). For particular applications, visualization of the NANOGOLD® directly may be desirable. Generally this requires very thin samples and precludes the use of other stains.

NANOGOLD® provides a much improved resolution and smaller probe size over colloidal gold conjugates. However, because NANOGOLD® is only 1.4 nm in diameter, it will not only be smaller, but will appear less intense than, for example, a 5 nm gold particle.With careful work, however, NANOGOLD® may be seen directly through the binoculars of a standard EM even in 80 nm thin sections.However, achieving the high resolution necessary for this work may require new demands on your equipment and technique.Several suggestions follow:

1. Before you start a project withNANOGOLD® it is helpful to see it so you know what to look for.Dilute the NANOGOLD® stock 1:5 and apply 4 ml to a grid for 1 minute.Wick the drop and wash with deionized water 4 times.

2. View NANOGOLD® at 100,000 X magnification with 10 X binoculars for a final magnification of 1,000,000 X.Turn the emission up full and adjust the condenser for maximum illumination.

3. The alignment of the microscope should be in order to give 0.3 nm resolution.Although the scope should be well aligned, you may be able to skip this step if you do step

4. Objective stigmators must be optimally set at 100,000 X.Even if the rest of the microscope optics are not perfectly aligned, adjustment of the objective stigmators may compensate and give the required resolution.You may want to follow your local protocol for this alignment but since it is important, a brief protocol is given here:
   1. At 100,000 X (1 X 10⁶ with binoculars), over focus, under focus, then set the objective lens to in focus.This is where there is the least amount of detail seen.
   2. Adjust each objective stigmator to give the least amount of detail in the image.
   3. Repeat steps a and b until the in focus image contains virtually no contrast, no wormy details, and gives a flat featureless image.

5. Now underfocus slightly, move to a fresh area, and you should see small black dots of 1.4 nm size.This is the NANOGOLD®.For the 1:5 dilution suggested, there should be about 5 to 10 gold spots on the small viewing screen used with the binoculars. Contrast and visibility of the gold clusters is best at 0.2 - 0.5 m defocus, and is much worse at typical defocus values of 1.5 - 2.0 m commonly used for protein molecular imaging.

6. In order to operate at high magnification with high beam current, thin carbon film over fenestrated holey film is recommended. Alternatively, thin carbon or0.2% Formvar over a 1000 mesh grid is acceptable.Many plastic supports are unstable under these conditions of high magnification/high beam current and carbon is therefore preferred.Contrast is best using thinner films and thinner sections.

7. Once you have seen NANOGOLD® you may now be able to reduce the beam current and obtain better images on film. For direct viewing with the binoculars reduction in magnification from 1,000,000 X to 50,000 X makes the NANOGOLD® much more difficult to observe and not all of the golds are discernable.At 30,000 X (300,000 X with 10 X binoculars) NANOGOLD® particles are not visible.It is recommended to view at 1,000,000 X, with maximum beam current, align the objective stigmators, and then move to a fresh area, reduce the beam, and record on film.

8. If the demands of high resolution are too taxing or your sample has an interfering stain, a very good result may be obtained using silver enhancement to give particles easily seen at lower magnification.

Silver Enhancement of Nanogold® ParticlesNANOGOLD® will nucleate silver deposition resulting in a dense particle 2-20 nm in size or larger depending on development time. Our LI SILVER silver enhancement system is convenient and not light sensitive, and suitable for all applications. Improved results in the EM may be obtained using HQ SILVER, which is formulated to give more controllable particle growth and uniform particle size distribution.⁵

Specimens must be thoroughly rinsed with deionized water before silver enhancement reagents are applied. This is because the buffers used for antibody incubations and washes contain chloride ions and other anions which form insoluble precipitates with silver. These are often light-sensitive and will give non-specific staining. To prepare the developer, mix equal amounts of the enhancer and initiator immediately before use. NANOGOLD® will nucleate silver deposition resulting in a dense particle 2-20 nm in size or larger depending on development time. Use nickel grids (not copper).

Silver enhancement is performed as follows:

1. Rinse with deionized water (2 X 5 mins).
2. OPTIONAL (may reduce background): Rinse with 0.02 M sodium citrate buffer, pH 7.0 (3 X 5 mins).
3. Float grid with specimen on freshly mixed developer for 1-4 minutes (HQ SILVER), or as directed in the instructions for the silver reagent.More or less time can be used to control particle size. A series of different development times should be tried, to find the optimum time for your experiment.
4. Rinse with deionized water (3 X 1 min).

Fixing with osmium tetroxide may cause some loss of silver; if this is found to be a problem, slightly longer development times may be appropriate.

NOTE: Treatment with osmium tetroxide followed by uranyl acetate staining can lead to much more drastic loss of the silver enhanced NANOGOLD®particles. This may be prevented by gold toning:⁶

1. After silver enhancement, wash thoroughly with dionized water.
2. 0.05 % gold chloride: 10 minutes at 4°C.
3. Wash with deionized water.
4. 0.5 % oxalic acid: 2 mins at room temperature.
5. 1 % sodium thiosulfate (freshly made) for 1 hour.
6. Wash thoroughly with deionized water and embed according to usual procedure.

Light Microscopy with Nanogold®Features labeled with NANOGOLD® will be stained black in the light microscope upon silver enhancement. Different development times should be tried to determine which is best for your experiment. A suitable procedure is given below.

Samples must be rinsed with deionized water before silver enhancement. This is because the reagent contains silver ions in solution, which react to form a precipitate with chloride, phosphate and other anions which are components of buffer solutions. The procedure for immunolabeling with NANOGOLD® and silver enhancement is given below.

1. Incubate with NANOGOLD® reagent diluted 1/40 - 1/200 in PBS-BSA or another buffer suitable for the system under study, for 1 hour at room temperature.
2. Rinse with PBS (3 X 5 min).
3. Postfix with 1 % glutaraldehyde in PBS at room temperature (3 mins).
4. Rinse with deionized water (3 X 1 min).
5. OPTIONAL (may reduce background): Rinse with 0.02 M sodium citrate buffer, pH 7.0 (3 X 5 mins).
6. Develop specimen with freshly mixed developer for 5-20 minutes, or as directed in the instructions for the silver reagent. More or less time can be used to control intensity of signal. A series of different development times may be used, to find the optimum enhancement for your experiment; generally a shorter antibody incubation time will require a longer silver development time.
7. Rinse with deionized water (2 X 5 mins).
8. The specimen may now be stained if desired before examination, with usual reagents.

PBS-BSA Buffer:

20 mM phosphate150 mM NaClpH 7.400.5% BSA0.1% gelatin (high purity)

Optional, may reduce background:0.5 M NaCl0.05% Tween 20PBS Buffer:

20 mM phosphate150 mM NaClpH 7.40

To obtain an especially dark silver signal, the silver enhancement may be repeated with a freshly mixed portion of developer.

References

1. Hainfeld, J. F, and Furuya, F. R., J. Histochem. Cytochem., 40, 177 (1992); Furuya, F. R., Hainfeld, J. F., and Powell, R. D., Proc. 49th Ann. Mtg., Micros. Soc. Amer.; Bailey, G. W., and Hall, E. L. (Eds.); San Francisco Press, San Francisco, CA,1991, p.286.

2. Tracz, E., Dickson, D. W., Hainfeld, J. F., and Ksiezak-Reding, H. Brain Res., 773, 33-44 (1997); Gregori, L., Hainfeld, J. F., Simon, M. N., and Goldgaber, D. Binding of amyloid beta protein to the 20S proteasome. J. Biol. Chem., 272, 58-62 (1997); Hainfeld, J. F.; Safer, D.; Wall, J. S.; Simon, M. N.; Lin, B. J., and Powell, R. D.; Proc. 52nd Ann. Mtg., Micros. Soc. Amer.; G. W. Bailey and Garratt-Reed, A. J., (Eds.); San Francisco Press, San Francisco, CA, 1994, p. 132.

3. Hainfeld, J. F., and Furuya, F. R.; in Immunogold-Silver Staining: Principles, Methods and Applications (M. A. Hayat, Ed.), CRC Press, Boca raton, FL., 1995: pp. 71-96.

4. Krenács, T., and Krenács, L.; in Immunogold-Silver Staining: Principles, Methods and Applications (M. A. Hayat, Ed.), CRC Press, Boca raton, FL., 1995: pp. 57-69.

5. Humbel, B. M.; Sibon, O. C. M.; Stierhof, Y.-D., and Schwarz, H.: Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and In Situ hybridization experiments; J. Histochem. Cytochem., 43, 735-737 (1995).

6. Arai, R., et al.; Brain Res. Bull.28, 343-345 (1992).

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Nanoprobes was founded in 1990 by Dr. James F. Hainfeld, along with a group of scientists who were also alumni of Brookhaven National Laboratory. The independent research facility they formed has allowed Dr. Hainfeld and his colleagues the freedom to pursue some of the greatest challenges in research: seeking cures for cancer and other diseases.

While part of the funding for these endeavors has come from research grants, another source had to be found, as the scientific community faces ever-increasing government cutbacks. To this end, Nanoprobes began to offer many of the nanoparticle technologies developed by its members for use in other scientists' research.