Colloidal Gold as a Cytochemical Markerin Electron Microscopy

Marc HorisbergerResearch Department, Nestié Products Technical Assistante Co., Ltd., La Tour-de-Peilz, Switzerland

Colloidal gold is orange to red, displays electron-opaque properties andis capable of strong emission of secondary electrons. As gold particlescan be produced in different sizes and can be labelled withmacromolecules which keep their specific properties, gold markers havefound ases in light and fluorescent microscopy, and many applicationsin scanning and transmission electron microscopy.

In biology, the most sophisticated analysis perform-cd in vitro will never suppress the urge of an in-vestigator to visualize the exact location of a cellcomponent.

From the time the Dutch naturalist Antonie vanLeeuwenhoek (1632-1723) made his first discoverieswith a simple instrument, microscopy has indeedproved to be a powerful tooi for understanding themicrocosm. Thanks to patient efforts and to an amaz-ing skill in using lenses, van Leeuwenhoek was able toobtain results which, at the time, were thought to betruly marvellous. His experiments became so popularthat it was fashionable in the upper class society ofDelft to Eind distraction, satisfaction and even joy inobserving nature through his primitive microscopes.These inner feelings are still strongly feit by modernresearchers using much more powerful instruments.

Clearly, van Leeuwenhoek was a self-taughtmicroscopist, but his observations led to manydiscoveries of great importante. He was the first toobserve protozoa, spermatozoids and red blood cells,and the first representation of a bacterium is to befound in one of his drawings of 1682.

The development of optical microscopes has con-tinued since that time, but the limits imposed by thewavelengths of visible light prompted the search forhigh resolution techniques. The first transmissionelectron microscopes having better resolution thanlight microscopes were built in 1932. Thirty yearslater, the scanning electron microscope was introduc-cd. Scanning electron microscopy (SEM) gives athree-dimensional quality to specimen images. Nor-mally, the instrument is operated by scanning, orsweeping, a very narrow beam of electrons back andforth across a metal-coated specimen, revealing itssurface features rather than its internal structure.

The possibility of localizing specific tissue com-ponents attracted the early microscopists, but it was

Raspail (1794-1878), a French botanist, pharmacist,microscopist and politician, who first used themicroscope to study the chemical reactions of tissuematerials. He is considered to be the founder ofhistochemistry and some of the reactions which hediscovered are still applied today. Over the years, alarge number of techniques has been developed toidentify, locate and quantify specific tissues and cellcomponents. Among the methods having a narrowspecificity, the use of fluorescent antibodies was in-troduced in 1941 by Coons et al. (1). A great advancewas then achieved at the ultrastructural level with thedevelopment of enzyme cytochemistry (2, 3, 4) andthe application of particulate markers, such as fer-ritin, conjugated to antibodies (5, 6). Ferritin is alarge molecule, of weight 800 000, with a proteinshell of about 12 nm outer diameter surrounding acore of ferric hydroxide phosphate containing morethan 2 000 atoms of iron. In the transmission electronmicroscope, the core of ferritin can be seen as a darkspot. Although the ferritin technique is widely usedand is a powerful tool in the hand of cytochemists, themethod is rather time-consuming and expensive.

A new metallic and particulate marker was in-troduced more recently, namely colloidal gold. Itsfirst application as a specific marker for transmissionelectron microscopy (TEM) was described in 1971(7). The gold method was further developed byHorisberger et al, in 1975 for SEM (8) and in 1979for fluorescent microscopy (9). Gold particle mappingby X-rays was reported in 1979 (10). The preparationof gold markers has been described in detail byHorisberger and Rosset (11) and by Geoghegan andAckerman (12), and the method has been discussed ingeneral articles (13, 14, 15). The interested readershould refer to these articles.

Dispersions in which the particles are large com-pared with the magnitude of ordinary molecules, but

90 Gold Bull., 1981, 14, (3)

still small enough to possess reasonable stability, arein general called colloidal dispersions, colloidal solu-tions or sols. The name colloidal was coined in 1861by the Scottish chemist Thomas Graham (1805-1869)from the Greek word for glue. The Italian EnricoSelmi was the first to give a precise description of col-loids in the years 1845 to 1860 and he built a theoryof which some aspects are still valid today. However,Selmi's work attracted less attention than Graham'sstudies which were widely circulated, and to this dayGraham has held the title of 'father of colloids'.

Colloidal GoldColloidal gold, prepared by reducing a dilute solu-

tion of gold chloride, is usually of a striking orange tored colour which it retains for many years. Althoughcolloidal gold was known to the alchemists of the 17thcentury, it was Michael Faraday (1791-1867) whofirst made a scientific study of its preparation and pro-perties. Some of Faraday's original gold dispersionsin water, prepared in 1857, are still preserved at theRoyal Institution in London. Faraday also discoveredthat addition of small amounts of electrolytes turnsthe colour of the sols from ruby to blue and coagulatesthem. More important, he demonstrated that theseeffects can be prevented by addition of gelatin andother macromolecules (16).

The facility with which its colour may be changedis one of the most curious properties of colloidal gold.No other hydrosol has been shown to change colourby coagulation, with the exception of that of silverunder certain conditions. This phenomenon has notyet received an adequate explanation.

Many varieties of colloidal gold are known. Theyhave been produced by reducing gold salts with atleast 50 mineral and organic reagents such asphosphorus, formaldehyde, ethanol, tannic acid,ascorbic acid and sodium citrate. Depending upon themethod used, the particles vary in size. Forcytochemical uses, gold colloids are prepared essen-tially by three procedures. The smallest particles, Au 5

— the subscript conveniently indicates the meandiameter of the particles in nanometres — are obtain-ed by the procedure of Faraday (16), boiling a dilutesolution of gold chloride in the presence ofphosphorus (7). Au, 2 particles are produced at roomtemperature by reducing gold chloride with sodiumascorbate (13, 15). Larger granules (Au 16 to Au 75) areproduced by boiling gold chloride in the presence ofdecreasing amounts of sodium citrate (17). While theformation of gold particles by the ascorbate method ispractically instantaneous at room temperature, withphosphorus and citrate the boiling solution turnsfaintly blue and then changes to red, orange or violet,depending upon the size of the particles. Thedevelopment of the colour can be just as fascinating

today as it must have been to the alchemists whose`potable gold' was also colloidal in character.

Preparation of Gold MarkersColloidal gold particles are surrounded by clouds of

ions, the presence of which is thought to be respon-sible for their stability. The particles carry a netnegative charge which causes mutual repulsion. Theaddition of electrolytes results in a compression of theionic double layer and a reduction of the radius ofrepulsive forces. As a result, gold sols coagulate. Thecoagulation process can be prevented by simply mix-ing a protein with a gold sol, thereby adding a protec-tive coat to the particles. While Faraday stabilizedgolds sols against coagulation by electrolytes withgelatin, Faulk and Taylor used antisera and thusobtained a specific immunocolloid (Au,) capable ofinteracting specifically with cell surface antigens (7).Following this initial work, many different re-searchers have prepared gold markers by labellinggold particles with a vide variety of macromoleculessuch as toxins, hormones conjugated to protein,polysaccharides, glycoproteins, protein A, enzymes,antisera, immunoglobulins, lectins and fluorescentprobes — reviews are presented in (13) and (15). Mostmacromolecules bound to gold particles show no ap-parent change in their specific bio-activity. Allavailable evidente indicates that macromolecules re-main firmly attached to gold particles, presumablythrough a non-covalent binding process (13, 14, 15).

The stabilization of colloidal gold by proteindepends upon a number of factors such as pH, theelectric charge of the molecule and its molecularweight (11 to 15). Once the optietal conditions forstabilizing a sol have been established by a simplecoagulation test with sodium chloride (7, 11), goldmarkers may be readily prepared on a large scale. Thecolloid is added to a sufficient amount ofmacromolecules dissolved in water or possibly in avery dilute sodium chloride solution. The colloid isfurther stabilized against the possible formation of ag-gregates by adding polyethylene glycol — the mosteffective being Carbowax 20-M of Union Carbide (8,13) — and centrifuged. By this step, the colloid is con-centrated and freed of unbound macromolecules.Finally, the colloid is suspended in a suitable buffercontaining polyethylene glycol. The whole procedurecan easily be completed in 3 to 4 hours. The stabilityof gold markers during storage at 4°C is generallyexcellent (15).

Applications of Gold MarkersAu, to Au„ particles have been used to mark

specimens either by the direct or the indirectmethods. In the former, specimens are directly in-cubated with the gold marker in a single step. In the

Gold Bull., 1981, 14, (3) 91

Fig. 1 Stereoscopie SEM micrographs of the yeast Schizosaccharornyces pombe. Thecells were marked for galactomannan by the indirect method. They were in-cubated with the Ricinus communis lectin specific for galactose. The divalent lectinbound to cell wall galactomannan was in turn marked with Au ss particles labelledwith a galactan (guaran) which reacted with the free lectin binding site. Cross-walls established by fission were practically not marked (single arrows). However,the growing ends reacted witti the marker (double arrow). The specimen was notcoated witli uwetal. The stereopairs are best viewed with an optical viewer. Alter-natively, witti a little deterinination one can sce the cell in three-dimensions withthe naked eye by allowing the right eye to sec only the right-hand figure and theleft only the left-hand figure, and by encouraging the images to becorne fused inthe brain (stereopsis). However, most people achieve stereopsis more easily withiuversion by crossing the eyes. The viewer holds a peneil hetween the figuren.While looking with both eyes at the tip of the pencil, he raises it towards his eyesuntil the two images fase. After (20)

latter, specimens are first incubated with a surfacespecific ligand, most often a lectin or an antibody, andthen in one or more subsequent steps, the goldmarker is attached to the bound ligand throughspecific interactions. Marking is achieved by agitatinggently unfixed or glutaraldehyde-fixed specimenswith an excess of gold marker. When marking isdense enough, it can be seen by means of lightmicroscopy as an orange-red surface coating (12). Themethod has also found application in fluorescentmicroscopy when gold particles are labelled with afluorescein (17) or a rhodamine (9) conjugate.

In SEM, the gold method has been used to mark avariety of cells such as yeast, red blood cells, platelets,hepatocytes and milk-fat globules (13, 15). One ad-vantage of SEM is that comparatively large areas canbe seen in detail. Contrary to other conventionalSEM markers, gold markers are good emitters ofsecondary electrons. This property enables theobserver to locate gold particles on a cell surfacewhich is not coated with a metal. At present, with thetype of instruments used, the most convenient size ofgold marker is approximately 50 nm, although par-ticles as small as Au22 have found uses (15).Stereomicrographs facilitate differentiation betweenindividual particles (Figure 1). Despite the simplikityof the method, SEM observations should be cor-roborated by TEM observations with smaller markerssince some cell surface binding sites may not be ac-

cessible to large size markers due to steric hindrance.In TEM, gold markers are readily identified by

their opacity to electrons and their characteristicshapes. The choice of the partiele size depends uponthe magnification used and consideration of the stericinaccessibility of binding sites. Au, and Au„ particleshave most commonly been used to mark virus,bacteria, yeast, plant cells and a great variety ofanimal cells (13, 15).

The method has found two applications of specialinterest in TEM, for which gold is unsurpassed byother particulate markers at present.

First, as the size of particles in monodisperse solscan be varied at will between 5 and 150 nm in meandiameter, gold markers are convenient for multiplemarking (10, 18). This technique permits the preciseevaluation of changes occurring on cell surfaces andthe study of the spatial location of receptors relative toeach other. In multiple marking experiments withgold particles, stereomicrographs must be taken toavoid false interpretation since apparently closelyassociated particles may be in different planes of thespecimen (Figure 2).

Secondly, the possibility of marking intracellularcomponents is another important application of col-loidal gold preparations, since many difficulties inachieving this goal are encountered using other par-ticulate markers. In contrast to ferritin conjugates,gold markers are easily recognized on thin sections to

92 Gold Bull., 1981, 14, (3)

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' •. —^.^— ^.. ^ _leiFig. 2 Stereoscopie TEM micrographs of triple-marked mouse embryofibroblasts. Binding sites (glycoproteins) for concanavalin A (Con A), soya bean(SBA) and wheat germ agglutiniu (WGA) were located by markingglutaraldehyde-fixed eells successively with Con A-Au 5 , SBA-Au17 and WGA-Au26. Soine SBA-Au17 particles are shown by the arrows. When the stereopair isexamined, inost of the markers are bound by spatially separated sites. Unpublish-ed documnents by Horisberger and Vonlanthen

which they bind nonspecifically to only a small ex-tent. Marking is achieved by floating ultrathin sec-tions on a droplet of gold marker. Again, bath thedirect and the indirect methods have been used(Figure 3). At present the most general method is theindirect protein A-gold method (19) which is based onthe ability of this protein to interact specifically witha wide variety of immunoglobulins from severalspecies.

As gold particles strongly absorb visible light at asingle peak of absorption (hmax 520 to 550 nm), a sim-

ple spectrophotometric method has been developed todetermine the number of particles bound per cell (11,13, 15). The latter depends upon a number of factorssuch as the Tonic strength of the buffer, the size of thegold particles and the method utilized to mark thespecimen (direct or indirect). As a rule, when the sizeof the marker increases, the number of bound par-ticles decreases, sometimes abruptly (13, 15). Thisindicates that binding sites close to the lipid bilayerare less accessible to large probes than receptorsextending from the eelt surface, or even that they are

Fig. 3 In an example of the direct method of using gold markers, thin sections of Candida uzilis were marked for mannonwith Con A-Aus (a) and anti-mannan antibodies-Aus (b). The indirect method is illustrated by sections which were in-cubated with anti-mannon antiserum and then with goat anti-rabbit IgG-Au$ (c) and protein A-Aus (d). Mannan was foundin the eelt walls and in some vacuoles. Although the results were. similar in all cases, the highest density of marking wasachieved by the protein A-gold mnethod. Unpublished observations by Horisberger and Vonlanthen

Gold Bull., 1981, 14, (3) 93

inaccessible to them. Therefore, the use of probes ofincreasing size appears to be a tooi for studying notonly the lateral, but also the longitudinal distributionof cell surface binding sites. However, since thenumber of labelled particles bound to a surface is lessthan that of the free molecules, the gold method doesnot allow an absolute quantification of specificsurface receptors. This restriction also appliesto other particulate markers.

ConclusionWhile the gold method has found some uses in

histochemistry, its main application is incytochemistry using both SEM and TEM. Themethod is general and its versatility resides in the

wide variety of macromolecules which can be adsorb-ed onto gold particles. Gold markers can be preparedrapidly and inexpensively, they show little non-specific adsorption and can be quantified by variousmethods. They are also useful for multiple markingexperiments. Finally, using the technique, in-tracellular antigens can be located on thin sections onwhich the gold particles can be clearly identified.

AcknowledgementsThe author thanks Mrs. J. Rosset for the scanning electron

micrographs, Ms. M. Vonlanthen for the transmission electronmicrographs, Mrs. Beaud for the photographic work and Pro-fessor B. Hirt (Swiss Institute for Experimental Cancer Research,Lausanne) for the gift of mouse embryo fibroblasts.

References1 A. H. Coons, H. J. Creech, R. N. Jones and E. Berliner, 1.

Imnunol., 1942, 45, 159-1702 P. K. Nakane and G. B. Pierce, 1. Histochem. Cytochem.,

1966, 14, 929-9313 S. Avrameas, Bull. Soc. Chim. Biol., 1968, 50, 1169.11784 L. A. Sternberger, P. H, Hardy, J. J. Cuculis and H. G.

Meyer, 1. Histochem. Cytochem., 1970, 18, 315-3335 S. J. Singer, Nature, 1959, 183, 1523.15246 S. J. Singer and A. F. Schick, 1. Biopltys. Biochem. Cytol.,

1961, 9, 519-5257 W. P. Faulk and G. M. Taylor, Inrrnunochemistry, 1971, 8,

1081 -10838 M. Horisberger, J. Rosset and H. Bauer, Experientia, 1975,

31, 1147-11489 M. Horisberger and M. Vonlanthen, Histochemistry, 1979,

64, 115-11810 L. C. Hoyer, J. C. Lee and C. Bucana, in `SEM /1979f111',

SEM Inc., AMF O'Hare, IL. 60666, pp. 629-636

11 M. Horisberger and J. Rosset, 7. Histochem. Cytochem.,1977, 25, 295-305

12 W. D. Geoghegan and G. A. Ackerman, 1. Histochem.Cytochem., 1977, 25, 1187-1200

13 M. Horisberger, Biol. Cel!,, 1979, 36, 253-25814 S. L. Goodman, G. M. Hodges and D. C. Livingston, in

SEM/1980/I', SEM Inc., AMF O'Hare, IL. 60666, pp.136-146

15 M. Horisberger, in `SEM /1981', SEM Inc., AMF O'Hare,IL. 60666, in prcss

16 M. Faraday, Ann. Plrys., 1857, 101, 38317 G. Frens, Nature Phys. Sci., 1973, 241, 20-2218 M. Horisberger and M. Vonlanthen, J. Microsc., 1979, 115,

97-10219 J. Roth, M. Bendayan and L. Orci, 1. Histochem. Cytochem.,

1978, 26, 1074-108120 M. Horisberger, M. Vonlanthen and J. Rosset, Arch.

Microbiol., 1978, 119, 107-111

The Development of Gold DrugsThree reviews have appeared in recent years, which in-

dicate that the development of gold drugs for use inmedicine may be entering a new and less empirical phase.They are the following: `The Biological Chemistry of Gold:a Metallodrug and Heavy-Atom Label with VariableValency' by P. J. Sadier of Birbeck College, University ofLondon, (Strttct. Bonding, 1976, 29, 171-214); `The Mam-malian Biochemistry of Gold: an Inorganic Perspective ofChrysotherapy', by C. F. Shaw III of the University ofWisconsin-Milwaukee, (Inorg. Perspect. Biol. Med., 1979, 2,287-355) and `The Chemistry of the Gold Drugs Used inthe Treatment of Rheumatoid Arthritis', by D. H. Brownand W. E. Smith of the University of Strathclyde, (Chem.Soc. Rev., 1980, 9, (2), 217-240).

Although gold is absorbed by some plants, it cannot nor-mally be detected in animal tissues and is not regarded as anessential element in living systems. Its administration istherefore akin to that of toxic elements such as mercury andbasically different from that of biologically used elementssuch as copper and iron.

Gold distributes widely in the body in which it undergoesa variety of reactions. The most important of these appearto be with thiols, and it undoubtedly exercises some of its

biological effects through such reactions and the disturb-ance which they cause to normal metabolism. Newknowledge and new techniques are making studies of thesereactions more effective. As is emphasized by Brown andSmith, however, gold is not applied in the form of ene drugonly and the in vivo reactions of compounds such astriethylphosphinegold chloride, Auranofin and sodiumaurothiomalate are likely to be quite different. They do notappear to produce a common metabolite on administrationand their clearance rates from particular tissues vary, aswell as their effects on enzymes. This makes the study of thefundament als of gold therapy particularly difficult.

Nevertheless, the fact that gold complexes are effectiveagainst such a widespread disease as rheumatoid arthritis,for which there is no acknowledged cure, makes their fur-ther study important. Although applications of these com-plexes have been increasing in recent years, their use is stillrestricted by the toxic effects which are often associatedwith their administration. There is no reason to believe,however, that their toxic and therapeutic effects arenecessarily linked and that research will not lead to more ef-fective complexes and/or to improved conditions of ad-ministration. W.S.R.

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