SYMPOSIUM |
Correspondence to: James F. Hainfeld, Biology Department, Brookhaven National Laboratory, Upton, NY 11973. E-mail: hainfeld@bnl.gov
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Summary |
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Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab' antibody fragments, making small and stable probes. Silver enhancement then makes these small gold particles easily visible by EM, LM, and directly by eye. Another advance is the combination of fluorescent and gold probes for correlative microscopy. Chemical crosslinking of gold particles to many biologically active molecules has made possible many novel probes, such as goldlipids, goldNiNTA, and goldATP. (J Histochem Cytochem 48: 471480, 2000)
Key Words: gold, immunogold, electron microscopy, gold labeling, gold clusters, immunocytochemistry, colloidal gold, Nanogold
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Colloidal Gold |
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Colloidal gold has been the label of choice for electron microscopy for some time, since it is made in convenient well-defined sizes and is rendered immunoreactive by adsorbing antibodies, as found by
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Gold Clusters |
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A newer gold technology has emerged based on the use of gold clusters, which are gold compounds with a core of multiple gold atoms, with the gold atoms at the surface covalently attached to organic groups. Undecagold, Au11(P(C6H5)3)7, was first described by McPartlin and the structure solved by X-ray diffraction (
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A larger gold cluster was subsequently developed (
In keeping with the objective of making the smallest immunoprobe for best penetration and highest density labeling, the Fab' antibody fragment was chosen. Because the Fab' fragment is one third the size of a full IgG molecule and because the gold cluster can be site-specifically attached to the end opposite the antigen-combining region to preserve immunoreactivity, a probe that is significantly smaller than any IgGcolloidal gold probe can be made (Fig 2).
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Discussion |
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Properties of Gold Clusters
What are the properties of these small gold cluster compounds and how do they differ from colloidal gold?
Stability.
The gold particle is covalently linked to the antibody or other molecule. They are therefore more stable than colloid gold immunoprobes where antibodies are simply adsorbed, and activity can be undiminished even after a year or more. In contrast, colloidal undergoes some antibody dissociation (
Penetration.
The small gold cluster (1.4-nm Nanogold) coupled to an Fab' fragment (5 nm) is smaller than an IgG (15 nm), giving excellent penetration into tissues (
Better Labeling of Antigens.
It has been recognized in many studies (
Better Resolution.
Gold clusters have even been used to mark specific sites of single biomolecules (
Peptide and Small Molecule Conjugates Possible.
Gold clusters can be covalently attached to almost any small molecule, including peptides (
Small and Uniform Size.
Because the clusters are actual compounds, they have a specific size (
No Aggregation.
The small gold clusters do not aggregate even over time, so conjugates that contain one gold cluster attached to one Fab' can be prepared (
Chromatographically Purified. Using gel filtration, it is possible to isolate monomeric Fab'single Nanogold conjugates and to eliminate excess gold and other species.
Use on Gels.
Conjugates are small enough to run on gels. The gold retards proteins by the approximate weight of the gold (~5 kD for undecagold and ~15 kD for Nanogold) (
There are, however, some disadvantages of the gold cluster approach. First, silver enhancement is needed for many applications in order to "grow" the small gold size to more visible sizes (1020 nm or larger). This is typically a simple procedure, however, just floating the grid on the developer for a few minutes. In addition, newer microscopes are able to visualize Nanogold without enhancement, such as on cell surfaces in the SEM. However, silver enhancement does add another variable, and achieving the desired mean product size may take several tries. In addition, if overdeveloped, some background appears. Second, the size distribution of metal particles after silver enhancement is larger than colloidal gold preparations with the same mean size. Finally, gold cluster conjugates are much more difficult to prepare than colloidal gold. Organic phosphines and gold clusters must be chemically synthesized and the gold and conjugates purified by several ultrafiltration and liquid chromatography runs.
Undecagold vs Nanogold vs Ultrasmall Gold
The larger Nanogold cluster (1.4-nm gold core) has several advantages over Au11 (0.8 nm): it is directly visible by TEM (and better SEMs) at high magnification, is stable in the beam, and it develops better with silver (
It is possible to make colloidal gold in very small sizes, 1.0 nm or less, and proteins are adsorbed to these particles in the same way as with larger colloidal golds. Unfortunately these "ultrasmall gold" preparations have several disadvantages (
Labeling Reactions
The linking arm on the gold can be made to specifically react with thiols (e.g., cysteine residues on proteins), using a maleimide group, or with amino groups using an N-hydroxysuccinimide (Fig 3). Other specific reactions can be used to link to sugar groups in glycoproteins. These form stable covalent bonds and permit useful conjugates to be made, even with molecules that do not adsorb to colloidal gold.
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Nanogold is available as reagents that react with free thiols or amines. These can be used to make conjugates with primary antibodies or other molecules. The procedure for labeling is quite simple, and consists of mixing the reactive gold reagent with the molecule to be labeled, then, after several hours, purifying the conjugate by gel filtration chromatography, ultrafiltration, or dialysis (to remove unreacted gold or unlabeled molecules). More specifically, the protocol for labeling is as follows:
An example is the labeling of an Fab' fragment, shown diagrammatically in Fig 4. An electron micrograph of Fab' fragments labeled with Nanogold (after column chromatography to remove excess Nanogold reagent) is shown in Fig 5.
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Stoichiometry of Labeling
With colloidal gold, it is difficult to quantitate the amount of antibody bound per gold particle by UV-Vis spectroscopy because the gold dominates the spectral absorption. However, gold clusters are smaller and one Nanogold at 280 nm absorbs 1.4-fold as much as a single IgG molecule. In addition, the gold absorbs in the visible spectrum, whereas most proteins do not. Therefore, a simple calculation based on a UV-Visible spectrum (or measurements at two points, such as 280 and 420 nm) will yield the ratio of gold to antibody. For successful antibody labeling with Nanogold, this is close to 1:1. A more detailed discussion of the equations to use is found in
Silver Enhancement
Silver enhancement, also called autometallography, is a process by which silver is deposited on the gold surface (
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Because osmium poststaining for EM can oxidize and reverse the silver staining, it was found that lower concentrations of OsO4 (0.1% for 30 min rather than 1%) does not affect the silver signal and still gives excellent staining (
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Electron Microscopy |
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Scanning Transmission Electron Microscopy (STEM)
The darkfield high-resolution STEM is one of the best instruments for visualizing small gold clusters clearly. It has been used in projects where single proteins or protein complexes have been labeled (-chain of fibrinogen mapped the locus of this site on the fibrinogen molecule (Fig 8).
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Labeling Peptides
Small peptides do not adsorb well to colloidal gold but can be covalently attached to gold clusters by reacting a cysteine -SH group, or an amino (-NH2) group (either the -amino or one from a lysine residue) (Fig 9). In one example, the 40-amino-acid amyloid ß-peptide, a peptide important in the development of Alzheimer's disease, was labeled with Nanogold (
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In another example, the 11-amino-acid peptide substance P (SP) was labeled with SulfoNHSNanogold (
TEM
Many examples have now been reported in the literature of Nanogold labeling in tissue sections in the TEM, using both pre-embedding (
Light Microscopy
Use of silver-enhanced gold gives punctate, high-resolution detail that stands out against H&E and other cell stains. Because the small gold cluster immunoprobes or Nanogoldstreptavidin penetrate into tissues up to 40 µm, this has led to many new applications for gold labeling for the LM. Although brightfield optics are usually adequate for viewing, reflected light (using crosspolarized epi-ilumination) can specifically identify the metal deposits because they repolarize the light upon reflection.
FluoroNanogold
For correlative light and electron microscopy, a dual label containing both a fluorophore and gold would be advantageous. The use of two separate immunolabels in parallel experiments (fluorescent in one and the gold in the other) has been unsatisfactory in many cases because different localizations are frequently seen. It also demands two preparations. Fluorescent and gold probes would also be useful for screening to see that labeling is successful at the LM level before proceeding (with the same sample) to the more extensive EM preparation. Another application of a dual probe is to follow a process in living cells, and when the fluorescent image indicates the time of interest, cells can be fixed and examined at the ultrastructural EM level. Combined fluorescent and colloidal gold probes have had limited success because gold particles strongly quench the fluorescence, and large gold particle sizes are worse. The quenching depends also on the distance between fluorophore and gold, so that they must be spaced apart at an appropriate distance.
Combined fluorescent and gold probes have been prepared by attaching both Nanogold and fluorescein (
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Two other reports in this issue detail applications of FluoroNanogold, and the reader is referred to them for further information and experimental details: T. Takizawa and J.M. Robinson, "FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy," and J.M. Robinson, T. Takizawa, and D.D. Vandré, "Enhanced labeling efficiency using ultrasmall immunogold probes: immunocytochemistry."
Metallosomes
Gold clusters, because they can be covalently linked with almost any other molecule, can be attached to phospholipids or fatty acids (
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In Situ Hybridization
Certain genes or sequences can be detected in cells, and this is important for both basic studies and pathology. For example, HPV-16 (human papilloma virus) is highly correlated with cervical cancer (
Gels and Blots
With silver enhancement, signals from immunogold targeting, for example, may be seen by eye on blots, as shown in Fig 14. Nanogold has shown to give particularly sensitive detection, rivaling or exceeding limits of fluorescent, radioactive, enzyme-colorometric, and chemiluminescent methods (
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Colloidal gold is generally too large to enter into polyacrylamide gels. However, gold clusters (undecagold ~5 kD and Nanogold ~15 kD) attached to proteins shift the weight by approximately that amount. This means that Coomassie blue staining can be used to visualize proteins, and silver staining will reveal the gold-labeled bands (using a parallel gel, or a lane cut lengthwise, half for Coomassie, half for silver) (
New Developments
Metal probes that can be covalently linked to biomolecules open up many possibilities for making novel probes for light and electron microscopy or other applications. Different molecules can be attached or different sized metal particles used, as well as a variety of metals in place of gold. Some of these are briefly described below.
NiNTANanogold
Cloned proteins frequently have a 6x-His tag (6 histidine tail) engineered in, which binds to a nickel column for one-step purifications from cell lysates. The nickel is held to the column material via the chelator NTA (nitrilotriacetic acid). This chelator has been attached to Nanogold, then charged with Ni2+ ions, so that the Nanogold then binds to 6x-His tagged proteins (Fig 15) (
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ATPGold
ATP or its analogues linked to gold clusters can be used in several ways. One is to label nucleotide binding proteins. Another is to serve as a substrate for a polymerase or DNA synthesizer to incorporate gold into nucleic acid probes. An ATPgold cluster conjugate has recently been described (
Larger Platinum and Palladium Clusters
These clusters are based on 1,10-phenanthroline ligands instead of the phosphines used in gold clusters. Clusters ~2 nm contain ~200 heavy atoms, and clusters 2.4 nm (Pd561) and 3.6 nm (Pd2057) have been described. The advantages of these structures include the use of a different metal for spectroscopic detection and larger sizes for better visibility (
ThiolGold
In addition to phosphine-stabilized gold particles, ligands bound to the gold core via a sulfur donor are also stable and may prove useful (
Tetra Iridium
Clusters with four iridium atoms have been synthesized and linked to antibodies, viruses (
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Gold Developer
Silver enhancement has proved to be quite useful to amplify the signal from small gold particles. In a similar fashion, gold can be catalytically deposited on the targeted gold particles (Fig 17). Gold enhancement has a number of potential advantages compared to silver. It can safely be used before osmium tetroxide staining (it is inert to osmium), lower backgrounds are observed in many cases and, for SEM, gold gives a significantly higher backscatter signal (
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Summary |
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Metal cluster labeling offers significant performance improvements over labeling with colloidal gold in the conventional manner, including more quantitative labeling and improved labeling of macromolecules and antigens in cells and tissues. The covalent crosslinking rationale and the ability to selectively modify the reactivity of metal cluster labels have enabled the preparation of new types of probes that are not feasible with colloidal gold. The development of combined fluorescent- and metal cluster-labeled probes has enabled more precise correlative microscopy, and the variety of different metals and cluster functionalities now available has significantly widened the applications of gold labeling.
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Footnotes |
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Presented in part at the New Frontiers in Gold Labeling Symposium, 5th Joint Meeting of the Japan Society of Histochemistry and the Histochemical Society, University of CaliforniaSan Diego, July 2326, 1998.
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Acknowledgments |
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Supported by the Office of Biological and Environmental Research of the US Department of Energy under Prime Contract No. DE-AC02-98CH10886 with Brookhaven National Laboratory, by National Institutes of Health Grant 2 P41 RR01777, and by NIH Small Business Innovation Research grants GM 48328, GM 49564, and GM 56090.
We wish to thank Dr Martha Simon, Ms Beth Lin, and Mr Frank Kito for STEM operation, and Dr Joseph Wall for helpful discussions. We are grateful to Carol M. R. Halsey, Vishwas Joshi, and Frederic Furuya for helpful experimental discussions, and to Gerhard W. Hacker for experimental data on gold enhancement and combined Cy3 and Nanogold probes.
Received for publication November 27, 1999; accepted December 1, 1999.
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