Review of Post-embedding Immunogold Methods for the Study of Neuronal Structures

The post-embedding immunogold (PI) technique for immunolabeling of neuronal tissues utilizing standard thin-section transmission electron microscopy (TEM) continues to be a prime method for understanding the functional localization of key proteins in neuronal function. Its main advantages over other immunolabeling methods for thin-section TEM are (1) fairly accurate and quantifiable localization of proteins in cells; (2) double-labeling of sections using two gold particle sizes; and (3) the ability to perform multiple labeling for different proteins by using adjacent sections. Here we first review in detail a common method for PI of neuronal tissues. This method has two major parts. First, we describe the freeze-substitution embedding method: cryoprotected tissue is frozen in liquid propane via plunge-freezing, and is placed in a freeze-substitution instrument in which the tissue is embedded in Lowicryl at low temperatures. We highlight important aspects of freeze-substitution embedding. Then we outline how thin sections of embedded tissue on grids are labeled with a primary antibody and a secondary gold particle-conjugated antibody, and the particular problems encountered in TEM of PI-labeled sections. In the Discussion, we compare our method both to earlier PI methods and to more recent PI methods used by other laboratories. We also compare TEM immunolabeling using PI vs. various pre-embedding immunolabeling methods, especially relating to neuronal tissue.

Determination of Gold Particle Characteristics for Sampling Protocol Optimisation

Sampling, sample preparation, and assay protocols aim to achieve an acceptable estimation variance, as expressed by a relatively low nugget variance compared to the sill of the variogram. With gold ore, the typical heterogeneity and low grade generally indicate that a large sample size is required, and the effectiveness of the sampling protocol merits attention. While sampling protocols can be optimised using the Theory of Sampling, this requires determination of the liberation diameter (dℓAu) of gold, which is linked to the size of the gold particles present. In practice, the liberation diameter of gold is often represented by the most influential particle size fraction, which is the coarsest size. It is important to understand the occurrence of gold particle clustering and the proportion of coarse versus fine gold. This paper presents a case study from the former high-grade Crystal Hill mine, Australia. Visible gold-bearing laminated quartz vein (LV) ore was scanned using X-ray computed micro-tomography (XCT). Gold particle size and its distribution in the context of liberation diameter and clustering was investigated. A combined mineralogical and metallurgical test programme identified a liberation diameter value of 850 µm for run of mine (ROM) ore. XCT data were integrated with field observations to define gold particle clusters, which ranged from 3–5 mm equivalent spherical diameter in ROM ore to >10 mm for very high-grade ore. For ROM ore with clusters of gold particles, a representative sample mass is estimated to be 45 kg. For very-high grade ore, this rises to 500 kg or more. An optimised grade control sampling protocol is recommended based on 11 kg panel samples taken proportionally across 0.7 m of LV, which provides 44 kg across four mine faces. An assay protocol using the PhotonAssay technique is recommended.

Detrital Gold as an Indicator Mineral

Detrital gold fulfils the criteria of chemical inertia and physical durability required by indicator minerals but it has not found wide application in this role because it may be formed in different deposit types. This problem is soluble, because the generic compositional features of hydrothermal gold differ according to mineralization environment. The wide distribution of gold as a minor component of mineralization where other commodities are the principle exploration target extends the potential of an indicator methodology based on detrital gold to beyond the search for gold itself. Here we highlight how distinctive gold compositional signatures derived from alloy composition and deposit- specific suites of mineral inclusions could contribute to exploration for Cu-Au porphyries, redox- controlled uranium mineralization and ultramafic-hosted PGE mineralization.Future refinement this approach will focus on establishing the spatial distribution of elements at trace levels within gold particle sections using ToF-LA-ICP-MS and application of Exploratory Data Analysis to the resulting data sets. This approach is in its infancy, but aims to develop a classification algorithm useful to researchers irrespective of their previous experience. A pilot study has that random forests provide the best approach to establishing gold particle origins.Supplementary material at https://doi.org/10.6084/m9.figshare.c.5625450

Evaluation of the contributions of gold derived from hypogene, supergene and surficial processes in the formation of placer gold deposits

Placer gold particles have traditionally been considered as either detrital products of weathering or authigenic minerals growing within placers. Recent advances in understanding of gold chemistry/bio-geochemistry demonstrate that gold growth in specific environments is plausible, but opinions differ on the importance of ‘new’ gold in the overall placer inventory. Here we draw upon visual inspection over 40,000 polished gold particle sections from locations worldwide to evaluate the implications of gold alloy composition and particle heterogeneity in determining the contributions of detrital and authigenic gold to fluvial placers. We conclude i. the detrital model of placer gold formation is widespread and demonstrable, ii. supergene gold may be a locally important constituent of fluvial placers, iii. gold-rich rims on placer gold particles comprise two distinct components: a surface micron-scale addition of pure Au and a tens- of- micron- scale inner rim formed by Ag depletion, iv. the importance to placer inventories of gold particle formation and modification by biogenic processes is considerably overstated.

A deep learning approach to identifying immunogold particles in electron microscopy images

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.

Laser Superficial Fusion of Gold Nanoparticles with PEEK Polymer for Cardiovascular Application

This paper analyses the possibility of obtaining surface-infused nano gold particles with the polyether ether ketone (PEEK) using picosecond laser treatment. To fuse particles into polymer, the raw surface of PEEK was sputtered with 99.99% Au and micromachined by an A-355 laser device for gold particle size reduction. Biomimetic pattern and parameters optimization were key properties of the design for biomedical application. The structures were investigated by employing surface topography in the presence of micron and sub-micron features. The energy of the laser beam stating the presence of polymer bond thermalisation with remelting due to high temperature was also taken into the account. The process was suited to avoid intensive surface modification that could compromise the mechanical properties of fragile cardiovascular devices. The initial material analysis was conducted by power–depth dependence using confocal microscopy. The evaluation of gold particle size reduction was performed with scanning electron microscopy (SEM), secondary electron (SE) and quadrant backscatter electron detector (QBSD) and energy dispersive spectroscopy (EDS) analysis. The visibility of the constituted coating was checked by a commercial grade X-ray that is commonly used in hospitals. Attempts to reduce deposited gold coating to the size of Au nanoparticles (Au NPs) and to fuse them into the groove using a laser beam have been successfully completed. The relationship between the laser power and the characteristics of the particles remaining in the laser irradiation area has been established. A significant increase in quantity was achieved using laser power with a minimum power of 15 mW. The obtained results allowed for the continuation of the pilot study for augmented research and material properties analysis.

Gold particle geomicrobiology: Using viable bacteria as a model for understanding microbe–mineral interactions

The biogeochemical cycling of gold has been proposed from studies focusing on gold particle morphology, surface textures and associated bacteria living on the surface of gold particles. Additionally, it has been suggested that metabolically active bacteria on particles catalyse gold dissolution and gold re-precipitation processes, i.e. fluid–bacterial–mineral interaction within microenvironments surrounding particles. Therefore, the isolation and characterisation of viable bacteria from gold particles can be used as a model to improve the understanding of bacterial–gold interactions. In this study, classical microbiology methods were used to isolate a gold-tolerant bacterium (Acinetobacter sp. SK-43) directly from gold particles. The genome of this isolate contained diverse (laterally acquired) heavy-metal resistance genes and stress tolerance genes, suggesting that gene expression would confer resistance to a wide range of potentially toxic metals that could occur in the surrounding microenvironment. The presence of these genes, along with genes for nutrient cycling under nutrient-limited conditions highlights the genomic capacity of how Acinetobacter sp. SK-43 could survive on gold particles and remain viable. Laboratory experiments demonstrated that this isolate could grow in the presence of soluble gold up to 20 μM (AuCl3) and that >50% of soluble gold was reduced upon exposure. Collectively, these results suggest that Acinetobacter sp. SK-43 (and presumably similar bacteria) could survive the cytotoxic effects of soluble Au from particles undergoing dissolution. This study provides comprehensive insight on the possible bacterial contributions to gold biogeochemical cycling in natural environments.

Chemical and physical heterogeneity within native gold: implications for the design of gold particle studies

Studies of populations of gold particles are becoming increasingly common; however, interpretation of compositional data may not be straightforward. Natural gold is rarely homogenous. Alloy heterogeneity is present as microfabrics formed either during primary mineralization or by modification of pre-existing alloys by chemical and physical drivers during subsequent residence in either hypogene or surficial environments. In electron-probe-microanalysis (EPMA)-based studies, the combination of Cu, Hg, and Pd values and mineral inclusion suites may be diagnostic for source style of mineralization, but Ag alone is rarely sufficient. Gold characterization studies by laser-ablation-ICP mass spectrometry linked to both quadrupole and Time-of-Flight (ToF-MS) systems show that only Ag, Cu, and Hg form homogenous alloys with Au sufficiently often to act as generic discriminants. Where present, other elements are commonly distributed highly heterogeneously at the micron or submicron scale, either as mineral inclusions or in highly localized, but low concentrations. Drawing upon our own data derived from individual inspection and analyses of approximately 40,000 gold particles from 526 placer and in situ localities worldwide, we show that adequate characterization of gold from a specific locality normally requires study of a minimum of 150 particles via a two-stage approach comprising spatial characterization of compositional heterogeneity, plus crystallographic orientation mapping, that informs subsequent targeted acquisition of quantitative compositional data by EPMA and/or laser-ablation ICP-MS methods. Such data provide the platform to review current understanding of the genesis of gold particle characteristics, elevating future compositional studies from empirical descriptions to process-focused interpretations.