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.     

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

DNA sequence mapping using electron microscopy

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.     

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.     

Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography

The localization of scarce antigens in thin sections of biological material can be accomplished by pre-embedment labeling with ultrasmall immuno-gold labels. Moreover, with this method, labeling is not restricted to the section surface but occurs throughout the section volume. Thus, when combined with electron tomography, antigens can be localized in three dimensions in relation to the 3D (three-dimensional) ultrastructure of the cell. However, for visualization in a transmission electron microscope, these labels need to be enlarged by silver or gold enhancement. The increase in particle size reduces the resolution of the antigen detection and the large particles obscure ultrastructural details in the tomogram. In this paper we show for the first time that these problems can be avoided and that ultrasmall gold labels can be localized in three dimensions without the need for gold or silver enhancement by using HAADF-STEM (high angular annular dark-field-scanning transmission electron microscopy) tomography. This method allowed us to three-dimensionally localize Aurion ultrasmall goat anti-rabbit immuno-gold labels on sections of Epon-embedded, osmium-uranium-lead-stained biological material. Calculations show that a 3D reconstruction obtained from HAADF-STEM projection images can be spatially aligned to one obtained from transmission electron microscopy (TEM) projections with subpixel accuracy. We conclude that it is possible to combine the high-fidelity structural information of TEM tomograms with the ultrasmall label localization ability of HAADF-STEM tomograms.     

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.     

Intracellular Localized Surface Plasmonic Sensing for Subcellular Diagnosis

This paper proposes a method for diagnosing intracellular conditions and organelles of cells with localized surface plasmonic resonance (LSPR) by directly internalizing the gold nanoparticles (AuNPs) into the cells and measuring their plasmonic properties through hyperspectral imaging. This technique will be useful for direct diagnosis of cellular organelles, which have potential for cellular biology, proteomics, pharmaceuticals, drug discovery etc. Furthermore, localization and characterization of citrate-capped gold nanoparticles in HeLa cells were studied, by hyperspectral microscopy and other imaging techniques. Here, we present the method of internalizing the gold nanoparticles into the cells and subcellular organelles to facilitate subcellular plasmonic measurements. An advanced label-free visualization technique, namely hyperspectral microscopy providing images and spectral data simultaneously, was used to confirm the internalization of gold nanoparticles and to reveal their optical properties for possible intracellular plasmonic detection. Hyperspectral technology has proved to be effective in the analysis of the spectral profile of gold nanoparticles, internalized under different conditions. Using this relatively novel technique, it is possible to study the plasmonic properties of particles, localized in different parts of the cell. The position of the plasmon bands reflects the interactions of gold nanoparticles with different subcellular systems, including particle-nucleus interactions. Our results revealed the effect of the different intracellular interactions on the aggregation pattern of gold nanoparticles, inside the cells. This novel technique opens the door to intracellular plasmonics, an entirely new field, with important potential applications in life sciences. Similarly, the characterization of AuNP inside the cell was validated using traditional methods such as light microscopy and scanning electron microscopy. Under the conditions studied in this work, gold nanoparticles were found to be non-toxic to HeLa (cervical cancer) cells.     

Silver enhancement of lectin-gold and enzyme-gold cytochemical labelling of eggs of the nematode Onchocerca gibsoni

Summary Wheat germ agglutinin—gold and chitinase—gold complexes were used to demonstrate the presence of chitin on the surfaces of eggs of the animal parasitic nematodeOnchocerca gibsoni. The gold complexes were enhanced by silver intensification and examined by light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Distinctive labelling of the egg surfaces was obtained with both probes in all three microscope modes. The results indicate that the small colloidal gold markers (3–10 nm) commonly used for high resolution TEM studies may be silver enhanced and also used for sensitive LM and SEM studies.     

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.     

Cytotoxicity,intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells

Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs.     

抗体和寡核苷酸双标记纳米金生物探针的制备及性能分析

基于纳米金粒子与抗体静电吸附作用,与硫醇修饰的寡核苷酸共价结合,建立一种新的双标记纳米金生物探针的制备方法.通过透射电镜（TEM）、紫外光谱、斑点免疫金渗滤法、免疫金银染色光镜观察法、荧光标记法等检测探针表征,及表面抗体活性情况和寡核苷酸的覆盖率,同时采用变性聚丙烯酰胺凝胶电泳（PAGE）检测寡核苷酸的存在.结果表明,纳米金粒子同时连接抗体和寡核苷酸后生物性能良好,且每个纳米金粒子（10±3）nm表面可覆盖寡核苷酸(92±20)条,双标记纳米金生物探针的制备具有简捷、稳定的特点.可作为一种新型探针应用于超微量蛋白质检测.     

Viral detection by electron microscopy: past, present and future

Viruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions.     

Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.     

Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.     

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.     

Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.     

Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles

The three-dimensional organization and fine structure of cytoplasmic components within whole non-embedded bovine aortic endothelial cells were examined during their attachment and spreading in tissue culture. Cells were cultured directly on Formvar-coated gold grids, fixed in glutaraldehyde and osmium tetroxide, critical point dried and examined by transmission electron microscopy (TEM) using stereoscopic methods, and by scanning electron microscopy (SEM). Reorganization of cytoplasmic structures during cell spreading occurred in four sequential stages: (1) spreading of the plasma membrane and unstructured cytoplasmic matrix; (2) spreading of cytoplasmic fiber systems (microtubules, microfilament bundles and microtrabecular system); (3) alignment of microfilament bundles and formation of radial tracts of microtubules; and (4) centripetal movement of organelles along radial tracts. These stages observed by TEM correlated with progressive degrees of cell flattening as visualized by SEM. These studies demonstrate that a characteristic reorganization of intracellular fiber systems and organelles accompanies the spreading of endothelial cells in culture.     

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.