Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery

Kinetics, biodistribution, and histological studies were performed to evaluate the particle‐size effects on the distribution of 15 nm and 50 nm PEG‐coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance‐scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15–30 min after injection. Rats were injected in the tail vein with PEG‐coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.     

Chiral 1,1'-binaphthyl-2,2'-dithiol-stabilized gold clusters: size separation and optical activity in the UV-vis

Gold particles covered with 1,1'-binaphthyl-2,2'-dithiol (BINAS) were prepared. Using size exclusion chromatography, it was possible for the first time to separate the sample into fractions with different sizes and colors. Transmission electron microscopy shows that the particles are very small, in the order of 1 nm or slightly above. The absorption spectra of the separated samples show rich structure. The particles show size-dependent optical activity in metal-based electronic transitions. The shape of both the absorption and circular dichroism spectra of one of the smallest fractions exhibits similarities with the spectra reported for Au 11 covered by 2,2'-bis(diphenylphosphino)-1,1'-biphenyl although the spectra are shifted to shorter wavelengths in the case of the dithiol. The anisotropy factors, Delta epsilon/epsilon of these particles are as large as 4 x 10(-3), which is larger than the values reported for gold particles stabilized by phosphines and water-soluble thiols. This indicates that BINAS is particularly well-suited to impart chirality on to gold particles.     

Comparison of detecting sensitivities of different sizes of gold particles with electron-microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model

The detecting sensitivities of different-sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha-ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 micron 2 nongranule area from that in 1 micron 2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha- and beta-ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha- and the beta-ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.     

Carbohydrate-directed synthesis of silver and gold nanoparticles: effect of the structure of carbohydrates and reducing agents on the size and morphology of the composites

A monosaccharide (β-d-glucose) and polysaccharide (soluble starch) were used as structure directing and subsequently stabilizing agents for the synthesis of spherical nanoparticles (NPs) and nanowires of silver and gold. Homogeneous monodispersed Ag(0) nanoparticles (Ag NPs) of 15 nm diameter were obtained when 10⁻⁴ M AgNO₃ precursor salt was reduced in starch (1 wt %)–water gel by 1 wt % β-d-glucose. For a second preparation the effect of reducing agents on the synthesis of Au(0) metallic nanoparticles (Au NPs) of 2 × 10⁻⁴ M concentration prepared in a β-d-glucose (0.03 M)–water dispersion was studied first in detail. Different equivalent amounts of NaBH₄ and a number of pH values were evaluated for the reduction of the Au salt HAuCl₄·3H₂O to obtain Au NPs. The type and the amount of reducing agent, as well as the pH of the solution was shown to affect the size and morphology of the NPs. NaBH₄ (4 equiv) produced the smallest (5.3 nm (σ 0.7)) metallic particles compared to larger particles (10.0 nm (σ 1.4)) when the salt was reduced by 1 equiv of NaBH₄. Addition of excess NaBH₄ caused the NPs to settle out as a precipitate forming a mesh or wire structure rather than monodispersed particles. Low pH (pH 6) resulted in incomplete reduction, while at pH 8 the salt was completely reduced. When the salt was reduced by NaOH at pH 8, the particles were larger (14.2 nm) and less homogeneous (σ 2.8) compared to those from NaBH₄ reduction.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Biorecovery of gold by Escherichia coli and Desulfovibrio desulfuricans

Microbial precipitation of gold was achieved using Escherichia coli and Desulfovibrio desulfuricans provided with H2 as the electron donor. No precipitation was observed using H2 alone or with heat-killed cells. Reduction of aqueous AuIII ions by both strains was demonstrated at pH 7 using 2 mM HAuCl4 solution and the concept was successfully applied to recover 100% of the gold from acidic leachate (115 ppm of AuIII) obtained from jewelry waste. Bioreductive recovery of gold from aqueous solution was achieved within 2 h, giving crystalline Au0 particles (20-50 nm), in the periplasmic space and on the cell surface, and small intracellular nanoparticles. The nanoparticle size was smaller (red suspension) at acidic pH (2.0) as compared to that obtained at pH 6.0 and 7.0 (purple) and 9.0 (dark blue). Comparable nanoparticles were obtained from AuIII test solutions and jewelry leachate.     

Determination of the size distribution of liposomes by SEC fractionation,and PCS analysis and enzymatic assay of lipid content

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85±32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone. Published: May 15, 2002.     

Phospholipid-stabilized Au-nanoparticles

This communication outlines a simple two-step approach of modification of 1 nm diameter Au nanoparticles using an aqueous solution of (1,2-dipalmitoyl-sn-glycero-3-phosphothio-ethanol) phospholipid (PL). Transmission electron microscopy as well as particle size analysis show that, as a result of PL reactions with Au particles, the initial Au nanoparticle size increases to 5 nm. Considering the size of the PL and their ability to form liposomes, 5 nm diameter spheres indicate that the PL bilayer was attached to the surface of Au particles and the PL-Au interactions are facilitated by the presence of thiol functionality. The change of surface electronic properties of PL-stabilized Au particles is manifested by the disappearance of the 217 and 290 nm absorbances due to 5d-6sp transitions in Au, which is likely attributed to the presence of S-H functionalities which increase the free electron density of the particle. As a consequence, two surface plasmons resulting from a collective oscillation of electrons in response to UV excitation disappear.     

Metal nanoparticle pollutants interfere with pulmonary surfactant function in vitro

Reported associations between air pollution and pulmonary and cardiovascular diseases prompted studies on the effects of gold nanoparticles (Au NP) on pulmonary surfactant function. Low levels (3.7 mol % Au/lipid, 0.98% wt/wt) markedly inhibited adsorption of a semisynthetic pulmonary surfactant (dipalmitoyl-phosphatidylcholine (DPPC)/palmitoyl-oleoyl-phosphatidylglycerol/surfactant protein B (SP-B); 70:30:1 wt %). Au NP also impeded the surfactant's ability to reduce surface tension (γ) to low levels during film compression and to respread during film expansion. Transmission electron microscopy showed that Au NP generated by a seed-growth method were spherical with diameters of ∼15 nm. Including palmitoyl-oleoyl-phosphatidylglycerol appeared to coat the NP with at least one lipid bilayer but did not affect NP shape or size. Similar overall observations occurred with dimyristoyl phosphatidylglycerol. Dipalmitoyl-phosphatidylglycerol was less effective in NP capping, although similar sized NP were formed. Including SP-B (1% wt/wt) appears to induce the formation of elongated strands of interacting threads with the fluid phosphatidylglycerols (PG). Including DPPC resulted in formation of aggregated, less spherical NP with a larger size distribution. With DPPC, strand formation due to SP-B was not observed. Agarose gel electrophoresis studies demonstrated that the aggregation induced by SP-B blocked migration of PG-coated NP. Migration was also influenced by the fluidity of the PGs. It is concluded that Au NP can interact with and sequester pulmonary surfactant phospholipids and, if inhaled from the atmosphere, could impede pulmonary surfactant function in the lung.     

Some viscoelastic properties of human erythrocyte spectrin networks end-linked in vitro

We have succeeded in making macroscopic networks of end-linked human erythrocyte spectrin. The network junctions were made using erythrocyte protein 4.1 irreversibly attached to 5 nm (diameter) colloidal gold particles. Rotary shadowing electron microscopy verifies that the protein 4.1-labelled colloidal gold particles bind only to the tail end of the spectrin molecules. Electron micrographs of protein 4.1-labelled colloidal gold particles incubated at 4 degrees C with spectrin dimers reveal that 1-5 spectrin dimers attach to each protein 4.1-labelled colloidal gold particle yielding a spider-like appearance of these complexes. Incubation with a low concentration of spectrin tetramers instead of dimers leads to extensive formation of spectrin microaggregates whereas use of spectrin concentrations higher than 3 mg/ml and a molar ratio between spectrin tetramers and protein 4.1/Au of 4 leads to formation of macroscopic spectrin networks. We have quantitated the viscoelastic properties of such end-linked macroscopic spectrin networks using a gravitational pendulum viscoelastometer. We find that in vitro end-linked spectrin networks can be described by linear viscoelastic theory. The dynamic storage modulus increases almost linearly with the spectrin-protein 4.1/gold particle concentration when the spectrin concentration exceeds about 3 mg/ml and the molar ratio between spectrin tetramers and protein 4.1/Au is 4. At a spectrin concentration of 6 mg/ml and the same ratio between spectrin and protein 4.1/Au, we find a dynamic storage modulus at low frequency of about 80 dyn/cm2. This is in adequate agreement with what is predicted by simple elastomer theory.     

Enhancing LSPR Sensitivity of Au Gratings through Graphene Coupling to Au Film

A particular interesting plasmonic system is that of metallic nanostructures interacting with metal films. As the localized surface plasmon resonance (LSPR) behavior of gold nanostructures (Au NPs) on the top of a gold thin film is exquisitely sensitive to the spacer distance of the film-Au NPs, we investigate in the present work the influence of a few-layered graphene spacer on the LSPR behavior of the NPs. The idea is to evidence the role of few-layered graphene as one of the thinnest possible spacer. We first show that the coupling to the Au film induces a strong lowering at around 507 nm and sharpening of the main LSPR of the Au NPs. Moreover, a blue shift in the main LSP resonance of about 13 nm is observed in the presence of a few-layered graphene spacer when compared to the case where gold nanostructures are directly linked to a gold thin film. Numerical simulations suggest that this LSP mode is dipolar and that the hot spots of the electric field are pushed to the top corners of the NPs, which makes it very sensitive to surrounding medium optical index changes and thus appealing for sensing applications. A figure of merit of such a system (gold/graphene/Au NPs) is 2.8, as compared to 2.1 for gold/Au NPs. This represents a 33 % gain in sensitivity and opens-up new sensing strategies.     

Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions

Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL.     

Laser exposure of gold nanorods can increase neuronal cell outgrowth

The usage of gold nanoparticles (Au NPs) in biological applications has risen significantly over the last 10 years. With the wide variety of chemical and biological functionalization available and their distinctive optical properties, Au NPs are currently used in a range of biological applications including sensing, labeling, drug delivery, and imaging applications. Among the available particles, gold nanorods (Au NRs) are particularly useful because their optical absorption can be tuned across the visible to near infrared region. Here, we present a novel application of Au NRs associated with low power laser exposure of NG108‐15 neuronal cells. When cells were irradiated with a 780 nm laser, the average number of neurons with neurites increased. A similar stimulatory effect was observed for cells that were cultured with poly‐(4‐styrenesulfonic acid)‐coated and silica‐coated Au NRs. Furthermore, when the NG108‐15 cells were cultured with both bare and coated Au NRs and then irradiated with 1.2–7.5 W/cm² at 780 nm, they showed a neurite length increase of up to 25 µm versus control. To the best of our knowledge, this effect has never been reported before. While the pathways of the stimulation is not yet clear, the data presented here demonstrates that it is linked to the absorption of light by the Au NRs. These initial results open up new opportunities for peripheral nerve regeneration treatments and for novel approaches to addressing central nervous system axons following spinal cord injury. Biotechnol. Bioeng. 2013; 110: 2277–2291. © 2013 Wiley Periodicals, Inc.     

Further characterization of locust low density lipophorin induced by adipokinetic hormone

This study was designed to resolve basic questions concerning the nature of low density lipophorin (LDLp) which is induced by adipokinetic hormone (AKH). For this purpose, lipophorin was fractionated by density gradient ultracentrifugation and each fraction containing lipophorin was analyzed for diacylglycerol and associated apolipophorin-III (apoLp-III). The diacylglycerol content of LDLp fractions increased significantly as the density of the fraction decreased (116 micrograms/100 micrograms protein at a high density to 209 micrograms/100 micrograms protein at a lower density). On the other hand, the content of diacylglycerol in each fraction of HDLp remained almost constant (33 micrograms/100 micrograms protein). It was also found that the number of apoLp-III molecules associated with LDLp increased as the density decreased (from 6.9 mol/mol LDLp to 13.2 mol/mol LDLp). However, electron microscopic observation showed that LDLp particles in each of the fractions were extremely heterogeneous in size with diameters of 29.4 +/- 6.8 nm, 27.1 +/- 5.5 nm, and 26.3 +/- 5.7 nm for low, medium, and high density fraction, respectively. HDLp particles were very homogeneous in size irrespective of the fraction (15.9 +/- 1.5 nm, 15.6 +/- 1.5 nm, and 15.6 +/- 1.3 nm for the respective fractions). A theoretical analysis based on all the experimental data strongly supports the hypothesis that the heterogeneity in the size of LDLp particles does not reflect different densities, but rather, heterogeneity is the result of intermolecular fusion between LDLp particles of the same density.     

Binding and internalization of gold-labeled IFN-gamma by human Raji cells

Binding and internalization of gold-labeled IFN-gamma (IFN-gamma/Au) by human Raji cells was examined by scanning and transmission electron microscopy. For SEM, visualization of gold particles was enhanced by the silver enhancement technique and by backscattered electron imaging. Binding studies revealed distinct labeling of microvilli-bearing cells after incubation with at least 10 U/ml IFN-gamma/Au, whereas cells with a smooth surface showed substantially lower labeling. After application of higher IFN-gamma (greater than 200 U/ml) concentrations, labeling intensity remained constant, which is consistent with the concentration of radiolabeled IFN-gamma required for saturating receptors on Raji cells. The specificity of IFN-gamma/Au binding was demonstrated by complete displacement with unlabeled IFN-gamma and by partial inhibition of labeling with a monoclonal anti-IFN-gamma R antibody. Thus, colloidal gold represents a valuable tag for visualizing the interaction of IFN-gamma with its receptor. Internalization of IFN-gamma/Au was initiated by accumulation of gold particles in coated pits which occurred within 10 min after warming of Raji cells. Additional incubation at 37 degrees C (up to 2 h) led to the appearance of gold particles in endocytic vesicles and lysosomes. Thus, our studies indicate that IFN-gamma/Au enters the Raji cells via the typical endocytotic pathway.     

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.     

The three-dimensional distribution of αA-crystalline in rat lenses and its possible relation to transparency

Lens transparency depends on the accumulation of massive quantities (600-800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated "fiber" cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ～2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (～3 nm and ～7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ～7.5 nm fitted the distances between the ～3 nm diameter gold conjugates. A Gaussian distribution centered at ～14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21-24 nm the distances between the larger particles. Independent of their diameters, tethers of 14-17 nm in length connected files of gold particles to thin filaments or clusters to ～15 nm diameter "beads." We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ～7 nm or αA-crystalline dimers spaced ～15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation.     

"Thiocyanate gold": small (2-3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy

Reduction of HAuCl4 by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation approximately 15%). The AuSCN sol forms protein-gold complexes. The amount of protein required to form an AuSCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates. By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing alpha-amylase in rat pancreatic acinar cells.     

One‐step synthesis of cationic gold nanoclusters with high catalytic activity on luminol chemiluminescence reaction

The present study reports a one‐step synthesis method for the preparation of cationic gold nanoclusters (Au NCs). Polyethyleneimine (PEI), a positively charged hyperbranched polyamine, was selected as the capping reagent. Glutathione showed a synergistic effect on the formation of the small size of cationic Au NCs. The prepared cationic Au NCs have a size less than 2 nm and carry a positive charge in solution with pH less than 11. The cationic PEI–Au NCs‐triggered luminol chemiluminescence (CL) reactions showed slow and intense CL profiles. The maximum CL intensity can be obtained within 10 min and the CL signal maintained almost the same within 30 min. A linear increase of CL intensity was observed in the presence of an increasing concentration of cationic Au NCs ranging from 0.030 μM to 15 μM. The linear response of the cationic Au NCs in the CL reaction and the glow‐type CL profile make the proposed CL reaction have broad application prospects in the field of biological analysis and CL imaging.