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Updated: September 19, 2005

N A N O P R O B E S     E - N E W S

Vol. 6, No. 9          September 19, 2005


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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Tetrairidium Carbonyl as a Microspectroscopy Probe

The carbonyl (C=O) group has an intense vibrational absorption band in the infrared (IR) or Raman spectrum. Metal carbonyl cluster compounds are thus easily detected by these techniques, and this provides a method for localizing targets by infrared or Raman microspectroscopy using metal carbonyls as detection labels.

Tetrairidium dodecacarbonyl, [Ir4(CO)12], has a versatile exchange chemistry with tris (aryl) phosphine ligands which allows the preparation of mixed carbonyl/phosphine compounds, and if modified phosphines are used to introduce cross-linkable reactive groups, may be converted to labels with advantages for several applications:

  • 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.

[Structure, STEM micrograph and IR image of Tetrairidum conjugate (81k)]

left: Schematic diagram of the tetrairidium cluster label. right: (upper) Scanning Transmission Electron Microscope (STEM) image of tetrairidium-Fab (full image width 128 nm), and (lower) Infrared (IR) image of tetrairidium-Fab captured by immobilized IgG (calculated as peak height ratio of 1990/2150 cm-1; the blue colored regions indicate highest concentration).

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).

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).

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).

More information:

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What is the Difference between HQ and LI Silver?

People frequently ask us which of our silver enhancers they should use for their particular application, what the differences between them are, and when it is better to use gold enhancement. There is considerable overlap between their uses, but they are based on different formulations, and differ significantly in their properties.

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:

Reagent: HQ Silver LI Silver
Development time: Fast: 1 to 8 minutes depending on desired final particle size. Normal: 15 to 40 minutes depending on desired detection sensitivity or final particle size.
Light sensitivity: Somewhat light sensitive. Use in darkroom under safelight or in dim or diffuse light. Light insensitive.
Advantages:
  • Highest size uniformity of developed particles.
  • Enlarges the greatest proportion of particles, giving the highest density of enlarged particles and the most quantitative labeling after silver enhancement.
  • Excellent ultrastructural preservation: ideal for use with fragile specimens or delicate techniques.
  • Highest sensitivity for optical and light microscopic detection.
  • Find exactly the right development time: slower development and light stability allows monitoring optically or by light microscopy
  • Low background.
  • Convenient two-component, non-viscous solution.
Applications:
  • Electron microscopy
  • Quantitative immunoelectron microscopic labeling, particle counting.
  • Immunogold labeling in delicate or less strongly fixed specimens.
  • Multiple labeling - uniform size allows easy differentiation.
  • Light microscopy: in situ hybridization and immunohistochemistry.
  • Immunoblotting and other blotting applications.
  • Enhancement of gold-labeled oligonucleotide and protein targets in gels and on transfer blots.
  • Biochips
  • Low-resolution electron microscopy, or electron microscopic screening.

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:

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Towards Bigger Nanogold®

One of our principal goals at Nanoprobes has been to extend the advantages of Nanogold® to larger gold labeling. We have previously described the preparation and use of a covalent 10 nm gold immunoprobe in which 10 nm gold particles, functionalized with novel alkanethiol ligands, were cross-linked to Fab' fragments via peripheral maleimide groups in the same manner as 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.

[Chromatographic separation and spectroscopic characterization of Au3nm-Fab'-Cy5 probes (77k)]

Separation of [Au3nm]-Fab-Cy5 by liquid chromatography over hydroxyapatite type 1, with UV/visible spectra for selected fractions. (red line shows gradient; scale shows fraction). Structure of the [Au3nm]-Fab-Cy5 is shown top right.

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:

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In Vivo Vascular Casting with Gold Nanoparticles

Accurately mapping and visualizing the blood circulation, in particular the blood supply to organs and tissues, is important for the diagnosis of many conditions and for evaluation of organ function. The vascular casting technique has permitted highly illuminating visualization; however, current methods require polymerization of a cast material within the animal being studied, and this is a terminal procedure. Therefore, in vivo processes cannot be monitored in the same animal over time to see real-time changes occurring normally or in response to environmental or biological factors such as diet, drugs, infection, tumor growth and other interesting processes.

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:

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Other Recent Publications

For those of you working with Fluorescence In Situ Hybridization (FISH), Ridderstråle and co-workers report a one-day procedure in the current issue of BioTechniques. The authors used a simplified procedure with microwave processing to overcome the effects of fixation. Using the commercially available probe mixture of HER-2 LSP (locus-specific probe) and CEP17 (centromeric probe for chromosome 17, alpha-satellite DNA), FISH was conducted on 4 micron thick formalin-fixed, paraffin-embedded tissue sections from 9 breast cancer and 7 endometrial cancer cases. Normal human lymphocytes and 4 breast cancer cell lines, all embedded in paraffin, were used as controls for hybridization and scoring efficiency. The microwave protocols produced results comparable to standard methods, and results from both treatments meet the criteria for HER-2 amplification status in BT-474 cells

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).

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:

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