N A N O P R O B E S     E - N E W S

Vol. 6, No. 9          September 19, 2005

• High-resolution electron microscopy. Tetrairidium compounds may be visualized in the Scanning Transmission Electron Microscope, or STEM, and because they are small, they label target sites with extremely high resolution. Therefore, they may be used for applications such as measuring the lengths of rigid organic molecules.

• Solving macromolecular structures through high-resolution electron microscopy combined with image analysis. Because it is so small, tetrairidium is able to access and bind to highly restricted sites, and can do so with less hindrance to the remainder of the molecule. The Journal of Structural Biology paper by Steven and co-workers demonstrates this application.

• As a size standard to determine the permeability of membranes or enclosed bodies, or to determine the size of holes or pores. In this role, it can be used as one member of a series of metal cluster compounds of different size but similar surface properties. Including its ligands, it is about 1.5 nm in diameter, while undecagold is 2.0 nm and Nanogold about 2.6 nm. Pore size or membrane permeability can be determined by which species pass through the pores into the interior and which do not.

At the recent Microscopy and Microanalysis meeting, we presented results describing the location of targets using tetrairidium-labeled antibody Fab' fragments, visualized both by scanning transmission electron microscopy (STEM) and infrared microspectroscopy. The tetrairidium cluster was converted to a maleimide and conjugated to freshly reduced goat anti-rabbit Fab' fragments using our established conjugation procedures. The Ir₄ labels are clearly visible in dark-field scanning transmission electron micrograph (STEM) of the Ir₄-labeled Fab conjugate. The Ir₄-F(ab) conjugate was then tested in a capture experiment, using 100 ng of covalently immobilized rabbit IgG on IR reflective slides. It was found that the captured Ir₄ labels could be localized at 10x10 micron resolution by FTIR microspectroscopy. Examples of STEM micrographs and IR localization are shown below.

These results demonstrate that these probes may be used for a novel correlative microscopic method: infrared microspectroscopy to determine the cellular or tissue distribution of specific chemical properties, and high-resolution electron microscopy to show the ultrastructural or even macromolecular localization of targets within specific structures or organelles. Infrared microspectroscopic imaging has been used to distinguish chemical differences between healthy and diseased tissues, and therefore we envisage the use of metal carbonyl cluster labels for rapid localization of diseased tissues. Subtraction of the Ir₄ label spectrum from that of localized targets may differentiate targets on the basis of their chemical properties, or provide information on these chemical properties that other methods, such as light or fluorescence microscopy, do not. The Ir₄ labels reported here also have active Raman vibrations. Raman microspectroscopy has also been used to distinguish healthy and diseased tissues; therefore, metal cluster carbonyl probes may also be useful as labels for Raman microspectroscopy or for correlative Raman microspectroscopy and electron microscopy.

Reference:

Joshi, V. N.; Ramamurthy, D.; Powell, R. D.; Furuya, F. R., and Hainfeld, J. F.: High Z Metal Carbonyls for Imaging and Microspectroscopy. Microsc. Microanal., 11 (Suppl. 2),; 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, 794CD (2005).

• Complete paper (courtesy of Cambridge University Press).

Reference - use of tetrairidium for STEM length measurement:

Furuya, F. R.; Miller, L. L.; Hainfeld, J. F.; Christopfel, W. C., and Kenny, P. W.: Use of Ir₄(CO)₁₁ to measure the lengths of organic molecules with a scanning transmission electron microscope. J. Amer. Chem. Soc., 110, 641-643 (1988).

• First page (PDF) (courtesy of the American Chemical Society).
• Article information (DOI) (courtesy of the American Chemical Society).

Reference - use of tetrairidium with image analysis for viral structure determination:

Cheng, N.; Conway, J. F.; Watts, N. R.; Hainfeld, J. F.; Joshi, V.; Powell, R. D.; Stahl, S. J.; Wingfield, P. E., and Steven, A. C.: Tetrairidium, a 4-atom cluster, is readily visible as a density label in 3D cryo-EM maps of proteins at 10 - 25 resolution. J. Struct. Biol., 127, 169-176 (1999).

• Abstract (courtesy of Science Direct).

More information:

• Complete paper
• Nanoprobes product and research applications: main page
• Guide to Gold Cluster Labeling
• Nanoprobes references, sorted by application

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What is the Difference between HQ and LI Silver?

First, the differences between the silver enhancers...

HQ Silver is formulated for the most uniform development, with the least perturbation of specimen ultrastructure. It contains a protective colloid, in order to control the rate of diffusion of the reactive species; this ensures that particles are enlarged uniformly, so that the enlarged particles have the least possible variation in size. It also permits the use of a near-neutral pH; this gives fast development, ensuring that the highest proportion of particles is developed; and because this reagent also has low ionic strength, it provides excellent ultrastructural preservation. This makes it ideal for electron microscopy applications where labeling density and size uniformity are critical, especially quantitative labeling applications, and also for delicate specimens with low fixation or sensitivity towards pH or ionic strength. However, this reagent is sensitive to sunlight or direct light, and therefore should be used in a darkroom with a safelight, or in diffuse light; for example, with the blinds closed or curtains drawn enough to leave sufficient light to see what you are doing.

LI Silver is formulated to give the most complete and specific development with the greatest degree of particle enlargement: it is formulated with components designed to minimize non-specific silver deposition or interaction with other features within the specimen. It requires a longer time than HQ Silver to achieve full development, and it is light insensitive and hence may be used in a fully lighted laboratory. It is intended for the highest staining and detection sensitivity in optical applications, including light microscopy, immunoblotting and gel staining. Because it develops more slowly and is light insensitive, it is ideally suited to applications where monitoring is needed; you can view the progress of the silver enhancement either by eye or in the light microscope, then stop when staining reaches the desired point either by rinsing with water, or treatment with freshly prepared 1 % sodium thiosulfate solution.

The key features and differences between these to reagents are summarized below:

But...we also offer gold enhancement, an alternative process in which gold, rather than silver, is deposited onto gold particles. The different properties and reactivity of gold, and its stability towards physiological ions such as halides and phosphates, means that gold enhancement has advantages for several applications. If you are planning to use the following techniques, you should consider using GoldEnhance

• Immunogold labeling in systems requiring physiological buffers that precipitate silver ions.

• Scanning electron microscopy (SEM) with backscatter detection. Gold has improved backscatter detection compared with silver, and therefore gold enhanced particles are better visualized by this method.

• Procedures where osmium tetroxide treatment is required and etching of silver-enhanced gold by osmium has been a problem; unlike silver, gold is not etched by osmium.

• Enhancement of gold-labeled specimens, such as cultured cells, on metal substrates. While silver can be deposited onto the metal substrates, gold usually is not.

• Some light microscopic applications, particularly in situ hybridization (ISH). Gold enhancement, used to visualize gold labeling as an ISH detection method, frequently produces lower background than silver enhancement, resulting in a cleaner or more specific signal.

Both silver and gold enhancement also work well with other immunogold reagents, including colloidal gold particles from other manufacturers. However, please note that both methods require the presence of gold nanoparticles. You cannot use silver enhancement on its own as a replacement for the formaldehyde-based "silver staining" methods: the chemistry of the process is different. In the formaldehyde silver staining system, the silver is deposited directly onto the biological molecule in question, and all the molecules on the gel are stained and visualized. In silver enhancement, the catalyst and nucleating center for silver deposition is an immunogold particle, and therefore silver enhancement will visualize only molecules linked to gold nanoparticle labels.

Please let us know if you need advice or assistance on selecting the best reagent for your application. We will be glad to advise.

More information:

• Silver enhancers: catalog information
• HQ Silver: product and technical information
• LI Silver: product and technical information
• Gold enhancement: general and catalog information
• Technical help: silver and gold enhancement
• References sorted by application: silver and gold enhancement

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Towards Bigger Nanogold®

3 nm gold is a particularly desirable size for an immunogold probe. A 3 nm gold Fab conjugate would be no larger than an IgG molecule, so it would penetrate as easily into cells and tissue sections, but would be significantly more easily visualized in the electron microscope than Nanogold, allowing imaging of its distribution in whole cells without the need for silver enhancement. We are therefore developing methods for preparing covalent 3 nm gold conjugates using particles cross-linked by coordinated organic ligands.

A major challenge in the preparation of such a probe is separating the conjugate from both unreacted gold and from unlabeled antibody, both because the components are similar in size, and because the UV/visible absorption from the gold is very large relative to that of the antibody; this prevents accurate calculation of the ratio of gold to antibody from the UV/visible spectrum as it is conducted for Nanogold. As we reported recently at Microscopy and Microanalysis 2005, in order to more accurately quantitate the antibody in preparations of 3 nm gold conjugates, we used Cy5-labeled Fab' fragments: Cy5 absorbs strongly at 650 nm, a wavelength at which absorbance due to gold is relatively low, and this absorbance may be used to more accurately quantitate the amount of Fab' than the protein absorption at 280 nm.

We used the 3nm gold conjugated Cy5-labeled Fab fragments to investigate different chromatographic and centrifugation separation methods. 3 nm gold particles, prepared by thiocyanate reduction and functionalized with a 10 : 1 mixture of hydroxyl-bearing and t-Boc-protected amino- alkanethiols, were deprotected with 0.3 M HCl in isopropanol), activated with sulfo-succinimidyl-4-N-maleimido-cyclohexane-1-carboxylate (sulfo-SMCC), and reacted in a 1 : 1 ratio with Cy5-labeled goat anti-rabbit Fab fragments, prepared by reduction of Cy5-labeled F(ab)2 with mercaptoethylamine hydrochloride (MEA) or dithiothreitol. Gold conjugates were separated by chromatography and by density gradient ultracentrifugation, and the composition of the separated species was examined spectroscopically.

Chromatographic separation was investigated using (a) hydroxyapatite type I, a combination gel filtration and hydrophobic interaction gel, eluted with a linear gradient of 0 to 100% buffer B (0.4 M sodium phosphate, pH 6.8, in 10% isopropanol/water), mixed with buffer A (5 mM sodium phosphate, pH 6.8, in 10% isopropanol/water), and (b) over Superose-6 gel filtration media eluted with 0.02 M sodium phosphate buffer with 0.15 M sodium chloride. Hydroxyapatite chromatography yielded two minor species, both eluted before the introduction of buffer B; UV/visible spectroscopy and scanning transmission electron microscopy (STEM) indicated that these contained mostly gold particles with little associated antibody. Two major peaks were eluted at close to 80 % and 95 % B: UV/visible spectroscopy and STEM indicated these contained principally unconjugated gold particles and partially labeled Fab respectively. The chromatographic separation and spectra of the separated species are shown below; the peak from fraction #29 shows the absorption due to the Cy5 at 650 nm, indicating the presence of antibody which would otherwise be very difficult to detect from these spectra. Gel filtration over Superose-6 resulted in only a single, broad peak, and UV/visible spectra and STEM of individual fractions indicated that this contained multiple overlapping species.

Density gradient ultracentrifugation is potentially a complementary method to gel filtration because the species are separated by density rather than size, so species containing gold particles should be separated from those of a similar size that do not due to the density of the gold. Separation over a 10 to 30 % sucrose gradient gave three layers; an upper band containing relatively little gold but significant amounts of antibody; a middle band containing both gold and antibody, consistent with a conjugate; and a lower band, containing little or no antibody.

Based on these results, hydroxyapapatite chromatography holds the most promise for conjugate separation from excess gold, and density gradient ultracentrifugation is most effective for separating conjugates from unlabeled Fab.

Reference:

Joshi, V. N.; Bhatnagar, A; Powell, R. D., and Hainfeld, J. F.: Towards Bigger Nanogold: Preparation of Covalent 3nm Gold Fab Probes. Microsc. Microanal., 11 (Suppl. 2),; 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, 1176CD (2005).

More information:

• Complete paper
• A covalent 10 nm gold immunoprobe: Our paper from Microsopy and Miroanalysis 1999
• Guide to Gold Cluster Labeling: separation of conjugates
• Other product and research applications

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In Vivo Vascular Casting with Gold Nanoparticles

The third new paper to be added to our web site this month, which was also presented at Microscopy and Microanalysis 2005, describes a novel application of gold nanoparticles for a potential in vivo vascular casting method. It has been found that gold nanoparticles, modified so as to be highly tolerated biologically, can provide enough contrast in the vascular system to enable the reconstruction of its architecture to high resolution using X-rays. Gold nanoparticles, 1.9 nm in diameter, were synthesized, and in preliminary toxicity studies, were found to be highly tolerated in test mice. After 2.7 g Au/kg, injected intraveneously, mice lived over one year without any signs of illness. This concentration produced an initial blood concentration of 36 mg Au/ml (i.e., the blood was 3.6% gold by weight).

With such a high level of gold, the blood vessels were clearly seen using a mammography unit, and blood vessels as small as 100 microns in diameter could be discerned. Vascular casting could then be accomplished by using a computed tomography (CT) or microCT scanner. The resolution limit of microCT machines is typically about 10 microns; however, the technique must allow for movement of the animal as well as the time needed to acquire such data, since scan times are much slower than for human CT scanners.

Post-mortum histology may also be used to further examine the distribution of the gold nanoparticles. An interesting tool is silver or gold enhancement, since it specifically visualizes the gold nanoparticles and can show their intracellular and extracellular biodistribution at any resolution necessary, from optical or light microscopy to high-resolution electron microscopy if localization within specific organelles is required.

Reference:

Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M., and Smilowitz, H. M.: In Vivo Vascular Casting. Microsc. Microanal., 11 (Suppl. 2),; 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, 1216CD (2005).

More information:

• Complete paper
• Nanogold® enhancement of cancer radiotherapy
• Nanogold: references, sorted by application
• Nanogold conjugates: general information
• Microscopy & Microanalysis 2005: meeting and abstract information

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Other Recent Publications

Reference:

Ridderstråle, K. K.; Grushko, T. A.; Kim, H.-J., and Olopade, O. I.: Single-day FISH procedure for paraffin-embedded tissue sections using a microwave oven. Biotechniques, 39, 316-320 (2005).

Shi, Taylor and group describe the standardization of a protein-protein embedding method for preparing controls for immunohistochemical experiments in this month's Journal of Histochemistry and Cytochemistry. Standardization is a particular challenge for immunohistochemistry (IHC) because the variability in sample processing and staining conditions and protocols can cause significant variation in the staining results. One approach to this problem is to use a control that is exposed to the same processing conditions in parallel with the specimen. This principle was previously adopted for the "Quicgel" method: this employs an artificial cell control block, which is added to the tissue cassette containing the clinical biopsy specimen and then subjected to routine fixation and embedding in paraffin. Although this provides a satisfactory reference material, it has not been widely accepted because it requires embedding an artificial cell line preparation with each specimen, and thus cannot be used for retrospective studies; it cannot control for the full range of different analytes (proteins) that the diagnostic pathologist wants to identify by IHC staining; and cell lines vary in behavior and are difficult to standardize. As an alternative, the authors used coated beads as the protein-embedding matrix for routine IHC on formalin-fixed paraffin-embedded tissue. Two experiments were described. In the first, beads coated with a goat anti-mouse antibody were bound with a monoclonal antibody to cytokeratin 7 at 4°C overnight in a cold room with an automatic shaker. Incubation was followed by three PBS washes, then biotinylated horse anti-mouse antibody was added and incubation continued under the same conditions for 3 hours. The beads, now coated with biotin-conjugated protein, were then fixed in 10% NBF for 20 min, mixed into 1% agarose gel in a small tube, and fixed in 10% neutral-buffered formalin (NBF) overnight. Blocks of agarose gel containing biotinylated protein-coated beads were subjected to the routine tissue-embedding procedure, including microwave antigen retrieval. In a second experiment, a purified S-100 protein was bound to the beads by incubation with a monoclonal mouse antibody against S-100 for 1 hour, followed after three PBS washes by a purified S-100 protein for 1 hour, followed by three PBS washes and the subsequent fixation, paraffin embedding, and antigen retrieval procedures described above. Slides prepared from these materials were incubated with the primary monoclonal antibody to S-100, followed by biotinylated anti-mouse antibody and the ABC incubation. Sections of a human melanoma were used as positive control for S-100. The beads demonstrated intense positive staining after antigen retrieval procedures, positive but less strong staining in the absence of antigen retrieval, and negligible staining in a negative control in which the ABC complex or primary antibody incubation was omitted. Based on these results, this approach holds promise as a versatile method for preparing IHC controls.

Reference:

Shi, S.-R.; Liu, C.; Perez J., and Taylor, C. R.: Protein-embedding Technique : A Potential Approach to Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections. J. Histochem. Cytochem., 53, 1167-1170 (2005).

• Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Dvorak reports some interesting - immunogold applications in her review of ultrastructural studies of human basophils and mast cells in the current Journal of Histochemistry and Cytochemistry. She describes the use of a novel enzyme affinity gold labeling method, actually developed some time ago, in which diamine oxidase-gold (DAO gold) is used to localize histamine. 15 nm colloidal gold was prepared according to the method of Frens: 4 ml of aqueous 1 % sodium citrate was added to 100 ml of boiling aqueous O.O1 % tetrachloroauric acid, allowed to boil for 5 min then cooled on ice. The pH of the resulting colloidal gold suspension was adjusted to 7 with 0.2 M potassium carbonate. 3 mg of DAO was dissolved in distilled water and placed in a polycarbonate ultrafuge tube with 10 ml of the gold suspension; the mixture was centrifuged at 25,000 rpm for 30 min, 4°C, in a Beckman ultracentrifuge with a #50.2 Ti rotor. The DAO-gold complex formed a red sediment that was carefully recovered and re-suspended in 3 ml 0.1 M PBS containing 0.02% polyethylene glycol, pH 7.6 (final concentration 1 mg DAO/ml). The resulting complex effectively localizes histamine, and was used extensively to study the localization of histamine and the mechanisms underlying its secretion and transport in mast cells and basophils.

References:

• Dvorak, A. M.: Ultrastructural studies of human basophils and mast cells. J. Histochem. Cytochem., 53, 1043-1070 (2005).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

• Dvorak A. M.; Morgan E. S.; Schleimer R. P., and Lichtenstein L. M.: Diamine oxidase-gold labels histamine in human mast-cell granules: a new enzyme-affinity ultrastructural method. J. Histochem. Cytochem., 41, 787-800 (1993).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

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