A Stereological Approach for Estimation of Cellular Immunogold Labeling and Its Spatial Distribution in Oriented Sections Using the Rotator

Particulate gold labeling applied to ultrathin sections is a powerful approach for locating cellular proteins and lipids on thin sections of cellular structures and compartments. Effective quantitative methods now allow estimation of both density and distribution of gold labeling across aggregate organelles or compartment profiles. However, current methods generally use random sections of cells and tissues, and these do not readily present the information needed for spatial mapping of cellular quantities of gold label. Yet spatial mapping of gold particle labeling becomes important when cells are polarized or show internal organization or spatial shifts in protein/lipid localization. Here we have applied a stereological approach called the rotator to estimate cellular gold label and proportions of labeling over cellular compartments at specific locations related to a chosen cell axis or chosen cellular structures. This method could be used in cell biology for mapping cell components in studies of protein translocation, cell polarity, cell cycle stages, or component cell types in tissues. (J Histochem Cytochem 57:709–719, 2009)     

Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations.     

Morphometric analysis of gold particles in low-label cell compartments.

Quantitative estimation of the binding of gold-conjugated ligands to various cell organelles has become a commonly used method to quantify the amount of ligand-binding sites associated with those organelles. However, often a small percentage of organelles is labeled or the density of gold labeling is low. We have defined the "gold-labeled region" as a zone that has a boundary defined by the localization of the outermost gold particles. Such a phenomenon was recently observed in a study of the internalization of gold-labeled native surfactant into lamellar bodies of cultured pulmonary type II cells. We estimated the size and density of gold-labeled regions in lamelar bodies using a simple stereological approach and demonstrated that the low percentage of gold-labeled organelles can be explained as a result of the probability of random selecting through the labeled areas. Our method, which permits use of transmission electron microscopy to calculate the true parameters of gold-labeled regions, can significantly facilitate analyses of ligand binding to various cell compartments.     

Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy

We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.     

A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.     

Quantifying immunogold localization on electron microscopic thin sections: a compendium of new approaches for plant cell biologists

A review is presented of recently developed methods for quantifying electron microscopical thin sections on which colloidal gold-labelled markers are used to identify and localize interesting molecules. These efficient methods rely on sound principles of random sampling, event counting, and statistical evaluation. Distributions of immunogold particles across cellular compartments can be compared within and between experimental groups. They can also be used to test for co-localization in multilabelling studies involving two or more sizes of gold particle. To test for preferential labelling of compartments, observed and expected gold particle distributions are compared by χ(2) analysis. Efficient estimators of gold labelling intensity [labelling density (LD) and/or relative labelling index (RLI)] are used to analyse volume-occupying compartments (e.g. Golgi vesicles) and/or surface-occupying compartments (e.g. cell membranes). Compartment size is estimated by counting chance events after randomly superimposing test lattices of points and/or line probes. RLI=1 when there is random labelling and RLI >1 when there is preferential labelling. Between-group comparisons do not require information about compartment size but, instead, raw gold particle counts in different groups are compared by combining χ(2) and contingency table analyses. These tests may also be used to assess co-distribution of different sized gold particles in compartments. Testing for co-labelling involves identifying sets of compartmental profiles that are unlabelled and labelled for one or both of two gold marker sizes. Numbers of profiles in each labelling set are compared by contingency table analysis and χ(2) analysis or Fisher's exact probability test. The various methods are illustrated with worked examples based on empirical and synthetic data and will be of practical benefit to those applying single or multiple immunogold labelling in their research.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

Distribution of anionic sites in gut tissue: an electron-microscopical study

Summary A new one-step incubation method using cationic gold colloid was applied to reveal anionic moieties in rat colonic mucosa. Gold particles were detected in all cellular nuclei, basement membranes, mast cell granules and collagen fibres, while the luminal surfaces of all vascular endothelial cells were devoid of gold label. Application of the method for detection of anionic domains under various conditions is discussed.     

Quantitative Assessment of Specificity in Immunoelectron Microscopy

In immunoelectron microscopy (immuno-EM) on ultrathin sections, gold particles are used for localization of molecular components of cells. These particles are countable, and quantitative methods have been established to estimate and evaluate the density and distribution of “raw” gold particle counts from a single uncontrolled labeling experiment. However, these raw counts are composed of two distinct elements: particles that are specific (specific labeling) and particles that are not (nonspecific labeling) for the target component. So far, approaches for assessment of specific labeling and for correction of raw gold particle counts to reveal specific labeling densities and distributions have not attracted much attention. Here, we discuss experimental strategies for determining specificity in immuno-EM, and we present methods for quantitative assessment of (1) the probability that an observed gold particle is specific for the target, (2) the density of specific labeling, and (3) the distribution of specific labeling over a series of compartments. These methods should be of general utility for researchers investigating the distribution of cellular components using on-section immunogold labeling. (J Histochem Cytochem 58:917–927, 2010)     

Immunocytochemical localization of heparan sulfate proteoglycan in human decidual cell secretory bodies and placental fibrinoid

A distinct ultrastructural feature of human decidual cells is the presence of membrane-bound secretory bodies, 0.3-0.5 micron in diameter, located within club-shaped processes at the cell periphery. These secretory bodies contain 30-60 nm electron-dense granules. Using specific antibody and the protein A-gold technique, we examined the localization of heparan sulfate proteoglycan in human decidual cells. Morphometric analysis of gold particles in cellular compartments was performed with a Zeiss Videoplan computer system. Immuno-gold staining was present in the decidual cell cytoplasm and the extracellular space, especially in the zone of the external lamina. Gold particles, indicating the locale of heparan sulfate proteoglycan, were concentrated over the electron-dense granular material within decidual secretory bodies contained in club-shaped processes at the cell periphery. Immunolabeling of placental fibrinoid was also observed. This report provides the first identification of a specific molecular constituent of decidual secretory bodies and indicates a role for these structures in secretion of the peri-decidual cell extracellular matrix.     

Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.     

A review of the study of protein secretion applying the protein A-gold immunocytochemical approach

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A-gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph-node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A-gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome-like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans-side and the trans-most rigid one. Quantitative evaluations of the intensities of labeling have allowed for 1) demonstration of the high specificity of the different labelings; 2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and 3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double-labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A-gold complexes formed by gold particles of different sizes. Protein A-gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high-resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A-gold technique, which in combination with other procedures constitutes a powerful method in cell biology.     

Nanoparticle based DNA biosensor for tuberculosis detection using thermophilic helicase-dependent isothermal amplification

The present study describes the development of a DNA based biosensor to detect Mycobacterium tuberculosis using thermophilic helicase-dependent isothermal amplification (tHDA) and dextrin coated gold nanoparticles (AuNPs) as electrochemical reporter. The biosensor is composed of gold nanoparticles (AuNPs) and amine-terminated magnetic particles (MPs) each functionalized with a different DNA probe that specifically hybridize with opposite ends of a fragment within the IS6110 gene, which is M. tuberculosis complex (MTC) specific. After hybridization, the formed complex (MP-target-AuNP) is magnetically separated from the solution and the AuNPs are electrochemically detected on a screen printed carbon electrode (SPCE) chip. The obtained detection limit is 0.01 ng/μl of isothermally amplified target (105 bp). This biosensor system can be potentially implemented in peripheral laboratories with the use of a portable, handheld potentiostat.     

Statistical evaluation of colocalization patterns in immunogold labeling experiments

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.     

Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures

Various methods for quantifying cellular immunogold labelling on transmission electron microscope thin sections are currently available. All rely on sound random sampling principles and are applicable to single immunolabelling across compartments within a given cell type or between different experimental groups of cells. Although methods are also available to test for colocalization in double/triple immunogold labelling studies, so far, these have relied on making multiple measurements of gold particle densities in defined areas or of inter-particle nearest neighbour distances. Here, we present alternative two-step approaches to codistribution and colocalization assessment that merely require raw counts of gold particles in distinct cellular compartments. For assessing codistribution over aggregate compartments, initial statistical evaluation involves combining contingency table and chi-squared analyses to provide predicted gold particle distributions. The observed and predicted distributions allow testing of the appropriate null hypothesis, namely, that there is no difference in the distribution patterns of proteins labelled by different sizes of gold particle. In short, the null hypothesis is that of colocalization. The approach for assessing colabelling recognises that, on thin sections, a compartment is made up of a set of sectional images (profiles) of cognate structures. The approach involves identifying two groups of compartmental profiles that are unlabelled and labelled for one gold marker size. The proportions in each group that are also labelled for the second gold marker size are then compared. Statistical analysis now uses a 2 × 2 contingency table combined with the Fisher exact probability test. Having identified double labelling, the profiles can be analysed further in order to identify characteristic features that might account for the double labelling. In each case, the approach is illustrated using synthetic and/or experimental datasets and can be refined to correct observed labelling patterns to specific labelling patterns. These simple and efficient approaches should be of more immediate utility to those interested in codistribution and colocalization in multiple immunogold labelling investigations.     

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.     

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.     

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.