Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species.

BACKGROUND: Timely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results. METHODS: Colloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera. RESULTS: AMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii. CONCLUSIONS: This study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Gold Particle Analyser: Detection and quantitative assessment of electron microscopy gold probes.

Gold particle probes are an essential electron microscopy tool to examine protein localisation, as well as protein trafficking. They can be introduced into living cells when conjugated to a protein that is endocytosed or to an antibody against a cell surface protein. Alternatively, gold particles can be introduced into fixed cells or tissue when conjugated to antibodies, immunoglobulin binding molecules or chemical probes applied to permeabilised samples or electron microscopy sections. Colloidal gold particles that have not been enlarged through chemical (gold or silver) enhancement are typically spherical and can be prepared in a range of specific sizes, allowing multiple proteins to be localised within a single sample. The typically homogeneous shape and size of the colloidal gold makes them ideal for computer assisted detection and analysis. Here we demonstrate a program developed to automatically identify two sizes of gold particle and perform a range of analyses that includes (i) distribution and cluster analysis; (ii) selection and analysis of gold particles allocated close to or either side of a membrane; (iii) measurement of organelle size; (iv) estimation of the number of gold particles within an aggregate and (v) the detection of chemically enhanced irregular sized and shaped gold particles. We show this easy-to-use program can greatly assist electron microscopists, to reliably and efficiently analyse gold particles within their images.

A simple and rapid immunochromatography test based on readily available filter paper modified with chitosan to screen for 13 sulfonamides in milk.

In this study, we developed a novel, simple, rapid, and low-cost colloidal gold-based immunochromatography method, with filter paper replacing nitrocellulose membrane as the substrate. To obtain adequately immobilized protein, chitosan was used to functionalize the filter paper. After conditions and parameters were optimized, the novel immunochromatography method was applied for detection of sulfonamide residues in milk. Quantitative detection was accomplished using a smartphone and Photoshop software (Adobe Inc., San Jose, CA), allowing us to screen 13 sulfonamides with a limit of detection ranging from 0.42 to 8.64 µg/L and recovery ranging from 88.2 to 116.9% in milk. The proposed novel method performed similarly to the conventional method that uses a nitrocellulose membrane as the transport medium, and it had lower cost and better usability because of the inexpensive and easily available filter paper.

Development and application of a colloidal gold test strip for the rapid detection of the infectious laryngotracheitis virus.

Infectious laryngotracheitis disease is an acute, highly contagious viral disease seriously affecting poultry industry worldwide. In this study, a rapid and simple immune colloidal gold test strip for detecting infectious laryngotracheitis virus (ILTV) was developed based on membrane chromatography with monoclonal antibodies (mAbs) against gJ protein of ILTV and systematically evaluated for the detection of ILTV from clinical samples. mAb 2D4 1D7 was conjugated with colloidal gold as the detector antibody on the test strip. Another mAb, 1D8 1G3, was used as the capture complex at the test line (T-line), and goat antimouse IgG antibody was used as the capture antibody at the control line (C-line). The colloidal gold test strip showed high specificity in the detection of ILTV, with no cross-reaction with other avian pathogens, including infectious bronchitis virus, infectious bursal disease virus, avian influenza virus, Newcastle disease virus, fowl adenoviruses, and Marek's disease virus. Besides, the detection limit of this method was as low as 60 ELD50/mL for the ILTV Wanggang strain. Furthermore, we evaluated its application in 260 clinical samples suspected of infection with ILTV. Results from the strip test were nearly identical with those from real-time PCR (coincidence rate 99.6%) and showed higher sensitivity than conventional PCR. All the results obtained in this study indicated that the colloidal gold test strip can be applied as a simple, rapid, sensitive, and specific diagnostic tool for the detection of ILTV, especially in resource-limited areas.

Development of an antigen specific colloidal gold immunochromatographic assay for detection of antibody to M. wenyonii in bovine sera.

The goal of this research was to develop a colloidal gold immunochromatographic strip test for detection of antibody to Mycoplasma wenyonii (M. wenyonii) in bovine using specific antigen. M. wenyonii was isolated from blood samples from the spontaneously infected cattle in Hebei province, China. Suspensions of the M. wenyonii antigenic proteins were prepared by freeze-thaw cycles and ultrasonication. Candidate antigens were screened with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The specific bands of the most antigenic proteins were excised from the gel and were purified by using a gel extraction kit. A colloidal gold immunochromatographic assay using the purified specific proteins as the coating antigen (sp-GICA) was developed for detection of antibody to M. wenyonii. Blood samples from cows in the field were tested for antibody to M. wenyonii by the sp-GICA strip and enzyme-linked immunosorbent assay (ELISA) simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the specific proteins bands with sufficient immunoreactivity have been identified. The apparent molecular weights of the proteins were 115 kDa and 60 kDa, respectively. The stability and reproducibility were quite excellent after the storage of the strip at room temperature for 5 months. This sp-GICA showed 95.48% (148/155), 92.86% (39/42) and 94.92% (187/197) in terms of specificity, sensitivity and accuracy compared to ELISA. The sp-GICA described here shows excellent agreement with ELISA and it is shown to be a simple, convenient, specific and highly sensitive assay for detection of serum antibodies to M. wenyonii.

A novel colloidal gold immunochromatography assay strip for the diagnosis of schistosomiasis japonica in domestic animals.

BACKGROUND: Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease. Therefore, development of a sensitive technique for the diagnosis of schistosomiasis in domestic animals is necessary. METHOD: A novel colloidal gold immunochromatography assay (GICA) strip was developed for detecting Schistosoma japonicum in domestic animals. The colloidal gold was conjugated with recombinant streptococcal protein G (rSPG). As the test and control lines, the schistosome soluble egg antigen and rSPG, respectively, were blotted on nitrocellulose membrane. RESULTS: The lowest detectable serum dilution was 1∶640 for schistosome-infected buffaloes. The cross-reaction rate of GICA was 14.29% with Paramphistomum sp. in buffaloes, 16.67% with Haemonchus sp. in goats, and 33.33% with Orientobilharzia sp. in goats. These results were slightly lower and similar to those obtained through ELISA. Moreover, the strips for detecting S. japonicum in mice, rabbits, buffaloes, and goats showed high sensitivity (100.00%, 100.00%, 100.00%, and 100.00%, respectively) and specificity (100.00%, 100.00%, 94.23%, and 88.64%, respectively). And the sensitivity or specificity of the GICA strips did not present any significant differences after storage for 12 months at room temperature. When compared with ELISA, the GICA strips exhibited similar sensitivity and specificity in the diagnosis of schistosomiasis in mice, rabbits, buffaloes, and goats. Besides, only 5 µl of serum are required for the test and the detection can be completed within 5 min. CONCLUSION: This study is the first to develop a GICA strip using gold-rSPG conjugate for the diagnosing of schistosomiasis in domestic animals, and preliminary results showed that the developed strip may be suitable for large-scale screening of schistosomiasis in endemic areas.

DNA-gadolinium-gold nanoparticles for in vivo T1 MR imaging of transplanted human neural stem cells.

The unambiguous imaging of transplanted cells remains a major challenge to understand their biological function and therapeutic efficacy. In vivo imaging of implanted cells is reliant on tagging these to differentiate them from host tissue, such as the brain. We here characterize a gold nanoparticle conjugate that is functionalized with modified deoxythymidine oligonucleotides bearing Gd(III) chelates and a red fluorescent Cy3 moiety to visualize in vivo transplanted human neural stem cells. This DNA-Gd@Au nanoparticle (DNA-Gd@AuNP) exhibits an improved T1 relaxivity and excellent cell uptake. No significant effects of cell uptake have been found on essential cell functions. Although T1 relaxivity is attenuated within cells, it is sufficiently preserved to afford the in vivo detection of transplanted cells using an optimized voxel size. In vivo MR images were corroborated by a post-mortem histological verification of DNA-Gd@AuNPs in transplanted cells. With 70% of cells being correctly identified using the DNA-Gd-AuNPs indicates an overall reliable detection. Less than 1% of cells were false positive for DNA-Gd@AuNPs, but a significant number of 30% false negatives reveals a dramatic underestimation of transplanted cells using this approach. DNA-Gd@AuNPs therefore offer new opportunities to visualize transplanted cells unequivocally using T1 contrast and use cellular MRI as a tool to derive biologically relevant information that allows us to understand how the survival and location of implanted cells determines therapeutic efficacy.

Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods.

An important tool in cell biology is the combination of immunogold labelling and transmission electron microscopy (TEM) by which target molecules (e.g. antigens) are bound specifically to affinity markers (primary antibodies) and then detected and localised with visualisation probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron-dense, punctate and available in different sizes whilst TEM provides high-resolution images of particles and cell compartments. By virtue of these properties, the combination can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labelling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labelling between compartments or groups of cells). The following approaches are possible: (i) A target molecule can be tested for preferential labelling by mapping the localisation of gold particles across a set of compartments. (ii) Data from wild-type and knockdown/knockout control cells can be used to correct raw gold particle counts, estimate specific labelling densities and then test for preferential labeling. (iii) The same antigen can be mapped in two or more groups of cells to test whether there are experimental shifts in compartment labelling patterns. (iv) A variant of this approach uses more than one size of gold particle to test whether or not different antigens colocalise in one or more compartments. (v) In studies involving antigen translocation, absolute numbers of gold particles can be mapped over compartments at specific positions within polarised, oriented or dividing cells. Here, the current state of the art is reviewed and approaches are illustrated with virtual datasets.

Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy.

Gold nanoparticles (AuNPs) have the potential to become a versatile biomarker. For further use of AuNPs labeled with functionalized molecules, their visualization in biological systems by routine laboratory tools such as light microscopy is crucial. However, the size far below the diffraction limit affords specialized parameters for microscopical detection, which stimulated the current study, aimed to determine from which size onward AuNPs, either in dispersion or cell-associated, can be reliably detected by standard confocal microscopy. First, gold colloids of size-restricted fractions are examined in dispersion. At a minimum particle size of 60 nm, detection appears to be reliable. Particle counts in dilution series confirm these results by revealing single particle detection of 60-nm colloids. Second, AuNPs are visualized and quantified in cells, which interestingly cause a phase shift in the reflection of AuNPs. Gold mass spectroscopy confirms the number of AuNPs counted microscopically inside cells. Furthermore, it demonstrates for the first time a very high diffusion rate of 15-nm particles into the cells. In conclusion, the results back the suitability of confocal microscopy for the quantitative tracking of colloidal and intracellular gold nanoparticles sized 60 nm.

Pre-embedding immunogold localization of antigens in mammalian brain slices.

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have been developed since the introduction of colloidal gold to immunocytochemistry. The two most widely used techniques, however, are based on transmission electron microscopy and consist of either immunolabeling before the specimens are embedded in resin (pre-embedding immunogold labeling) or immunolabeling after embedding in resin (post-embedding immunogold labeling). The following protocol describes a pre-embedding procedure that gives reliable results with all antibodies that produce adequate staining as observed with a light microscope. This procedure results in almost perfect preservation of the ultrastructure. The procedure employs thick sectioning using a vibratome, permeabilization of membranes with Triton X-100, and immunolabeling with fluorescently conjugated Nanogold antibodies, followed by gold enhancement and embedding for electron microscopy. We also discuss some limitations inherent to pre-embedding immunogold labeling.

Use of immunogold with silver enhancement.

Although gold particles are readily detectable by transmission electron microscopy, they can be difficult to visualize by bright-field light microscopy. However when the gold is silver-enhanced it is easy to see. During silver-enhancement, the colloidal gold serves as a nucleation site for the deposition of metallic silver. The enhancing solutions are physical developers that contain both silver ions and a reducing agent, buffered to an acid pH. The silver-enhancement method has also been used successfully to enlarge small-diameter gold particles for visualization by scanning electron microscopy. Silver-enhancement has been applied to a wide variety of tissues and antigens for both light and scanning electron microscopy.

Colloidal gold/streptavidin methods.

Biotin-avidin detection systems are widely used in both immunocytochemistry and molecular biology. They take advantage of the high affinity of biotin, a low-molecular-weight vitamin, for avidin, an egg-white protein. Because of the problem of nonspecific binding of avidin, streptavidin has largely replaced avidin for immunocytochemical procedures. Streptavidin/colloidal gold-biotin detection systems for electron microscopy are most commonly used in postembedding immunocytochemistry. Usually, the primary antibody is unlabeled, the secondary antibody is biotinylated, and the colloidal gold is conjugated to streptavidin. In certain applications, the primary antibody may be biotinylated and no bridging antibody is needed. This chapter details the use of streptavidin-gold for postembedding labeling.

Postembedding labeling methods.

Since it was first introduced, postembedding immunogold labeling has become the most widely used method of immunolabeling for electron microscopy. For postembedding labeling, samples are first fixed, embedded, and sectioned. All immunostaining is performed on sections mounted on grids. The immunostaining may be done with a direct labeling technique with the primary antibody conjugated to colloidal gold, or by an indirect method where the primary antibody is unlabeled and the gold is conjugated to a secondary or tertiary antibody, protein A, protein G, etc. Colloidal gold-antibody conjugates are also widely used, especially in indirect immunocytochemical methods. This chapter gives the standard method for post embedding labeling as well as one in which the cell membranes are enhanced.

Pre-embedding labeling methods.

Colloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been largely restricted to labeling cell-surface antigens for scanning or transmission electron microscopy or for tracing endocytic pathways in living cells. One nanometer gold conjugates that do penetrate cells and tissues much more readily have also been used successfully to immunolabel intracellular structures. For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This chapter provides methods for pre-embedding staining with unconjugated primary antibody or with primary antibody conjugated to colloidal gold. The use of colloidal gold for tracing endocytic pathways is also given.

A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs.

Detecting, localising and counting ultrasmall particles and nanoparticles in sub- and supra-cellular compartments are of considerable current interest in basic and applied research in biomedicine, bioscience and environmental science. For particles with sufficient contrast (e.g. colloidal gold, ferritin, heavy metal-based nanoparticles), visualization requires the high resolutions achievable by transmission electron microscopy (TEM). Moreover, if particles can be counted, their spatial distributions can be subjected to statistical evaluation. Whatever the level of structural organisation, particle distributions can be compared between different compartments within a given structure (cell, tissue and organ) or between different sets of structures (in, say, control and experimental groups). Here, a portfolio of stereology-based methods for drawing such comparisons is presented. We recognise two main scenarios: (1) section surface localisation, in which particles, exemplified by antibody-conjugated colloidal gold particles or quantum dots, are distributed at the section surface during post-embedding immunolabelling, and (2) section volume localisation (or full section penetration), in which particles are contained within the cell or tissue prior to TEM fixation and embedding procedures. Whatever the study aim or hypothesis, the methods for quantifying particles rely on the same basic principles: (i) unbiased selection of specimens by multistage random sampling, (ii) unbiased estimation of particle number and compartment size using stereological test probes (points, lines, areas and volumes), and (iii) statistical testing of an appropriate null hypothesis. To compare different groups of cells or organs, a simple and efficient approach is to compare the observed distributions of raw particle counts by a combined contingency table and chi-squared analysis. Compartmental chi-squared values making substantial contributions to total chi-squared values help identify where the main differences between distributions reside. Distributions between compartments in, say, a given cell type, can be compared using a relative labelling index (RLI) or relative deposition index (RDI) combined with a chi-squared analysis to test whether or not particles preferentially locate in certain compartments. This approach is ideally suited to analysing particles located in volume-occupying compartments (organelles or tissue spaces) or surface-occupying compartments (membranes) and expected distributions can be generated by the stereological devices of point, intersection and particle counting. Labelling efficiencies (number of gold particles per antigen molecule) in immunocytochemical studies can be determined if suitable calibration methods (e.g. biochemical assays of golds per membrane surface or per cell) are available. In addition to relative quantification for between-group and between-compartment comparisons, stereological methods also permit absolute quantification, e.g. total volumes, surfaces and numbers of structures per cell. Here, the utility, limitations and recent applications of these methods are reviewed.

Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes.

Translocation of inhaled ultrafine particles from the lungs into the blood may impair cardiovascular function. We administered ultrafine (20-nm) and fine (200-nm) gold colloid or fluorescein-labeled polystyrene particles to mice intratracheally and examined their localization in the lung and extrapulmonary organs. Fifteen minutes after instillation, dispersed and agglomerated 20-nm gold colloid particles were observed on the surface of endothelial cells, on the alveolar surface, in endocytotic vesicles of alveolar epithelial cells, and in the basement membrane of the lung. A small but noteworthy amount of gold was detected in the liver, kidney, spleen, and heart by inductively coupled plasma-mass spectrometry. After administration of 20- or 200-nm fluorescent particles, free particles were detected infrequently in blood vessels, on the endocardial surface, and in the kidney and liver only in the mice that received 20-nm particles, whereas phagocytes containing 20- or 200-nm particles were found in the extrapulmonary tissues. Fluorescent particle-laden alveolar macrophages administered intratracheally translocated from alveoli to extrapulmonary organs via the blood circulation. Thus, small amounts of ultrafine particles are transported across the alveolar wall into the blood circulation via endocytotic pathways, but particle-laden alveolar macrophages translocate both ultrafine and fine particles from the lungs to the extrapulmonary organs.

Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of enrofloxacin residues.

A rapid immunochromatographic lateral flow test strip of competitive format has been developed using a gold-conjugated monoclonal antibody for the specific determination of enrofloxacin (ENR) residues in chicken muscles. For this purpose, a specific monoclonal antibody (mAb) for ENR was generated and characterized. The mAb showed negligible cross-reactivity with other related compounds. Using ENR standards prepared in chicken muscle extracts from 0 to 24.3 ng/mL (microg/kg), the method indicated that the detection limit of the test strip, as measured in a strip scanner, was as low as 0.138 microg/kg of ENR and the half-maximal inhibition concentration (IC(50)) was 0.935 microg/kg. For samples spiked at 10, 20, and 30 microg/kg, the recovery was between 85.3 and 96.1% and the coefficient of variation [CV (%)] was between 4.5 and 7.91%. Parallel analysis of muscle samples from chickens fed ENR showed good comparable results obtained from the test strip and LC-MS. Each test requires 5-10 min. The data indicate that the method has high sensitivity, specificity, and the advantages of simplicity and speed of performance. Therefore, the test strip provides a useful screening method for quantitative, semiquantitative, or qualitative detection of ENR residues in chicken muscles.

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance.

To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 microg kg(-1), respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L(-1) phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 microg kg(-1) in liver and 2 microg kg(-1) in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r(2)) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 microg kg(-1)).

Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying).

In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume-occupying) membrane. However calculated, the LDs over the chosen (membrane and organelle) compartments are used to obtain observed and expected gold particle counts. The observed distribution is determined simply by counting gold particles associated with each compartment. Next, an expected distribution is created by randomly superimposing test points and counting those hitting each compartment. LDs of the chosen compartments are used to calculate RLI and chi-squared values and these serve to identify those compartments in which there is preferential labelling. The methods are illustrated by synthetic and real data.

Size and shape separation of gold nanoparticles with preparative gel electrophoresis.

We employed agarose gel preparative electrophoresis to separate gold nanoparticles based on size, shape, and charge. The separating technique was first demonstrated by size separation of 5 nm, 15 nm, and 20 nm spherical gold nanoclusters; and further evidenced through the purification of crude 15 +/- 2.7 nm nanoclusters to nanoclusters that were 15 +/- 0.4 nm. The ability to separate gold nanoparticles by shape was also shown by the purification of a mixture of gold spheres, plates, and long rods.