Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labelling multiple antigens on ultra-thin sections

Summary The preparation of a protein A-gold complex (pAg₃) using 3 nm gold particles and its application for labelling of intracellular antigens on thin sections is reported. The 3 nm gold particle is the smallest metal particle currently available for cytochemistry and permits a higher resolution of the pAg technique. Furthermore, it can be used in double labelling experiments in conjunction with a pAg complex prepared from 15 nm gold particles. For double labelling, the pAg₃ complex must be used for staining of the first antigen since otherwise a non-specific co-labelling of the two pAg complexes results.     

Metallosomes.

Structures and ordered arrays containing organometallic particles have potential application in nanofabrication, smaller computer components, optical devices, sensors, and membrane probes and as detection agents. Here, we describe construction of gold clusters covalently attached to lipids and their use in forming typical lipid structures: micelles, liposomes ("metallosomes"), and sheets on an air-water interface. Two sizes of gold clusters were used, undecagold, with an 11-gold atom core 0.8 nm in diameter, and the larger Nanogold, with a 1.4-nm gold core. The morphology of the structures formed was determined by electron microscopy at a resolution at which single gold-lipid molecules were visualized. Further modification by additional catalytic metal deposition enhanced detectability. The approach is flexible and permits a wide variety of metal particle structures to be created using known lipid structures as templates. Additionally, these gold-lipids may serve as useful membrane labels.     

Direct detection of immunogold reactions by real-time video microscopy

Summary Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

Direct detection of immunogold reactions by real-time video microscopy.

Video-enhanced microscopy allows the detection and tracking of individual colloidal gold particles. The analysis of immunogold reactions can also be conducted as a function of time and thus allows the study of dynamic events in living cells. The direct visualization in real time is reported of the reaction of immunogold particles with a surface antigen. This time-resolved immunocytochemistry was achieved by continuous observation of living cells infected with a virus (respiratory syncytial virus) following their incubation with colloidal gold (30 nm) coated with antiviral antibodies. The progress of the immunoreaction was visualized as a sequential deposition of individual gold granules on the viral particles until saturation was reached after 60 min. Binding of colloidal gold was an irreversible event as no elution or dislocation of surface-bound granules took place. Comparative imaging of colloidal gold particles by electron microscopy and by video microscopy demonstrated that the video-imaged immunoreactions represented events involving single gold particles; their signal was sometimes clearly enhanced by secondary depositions taking place in close proximity, i.e. at a distance below the lateral resolution of the light microscope. Our experiments demonstrate that video-enhanced microscopy provides a powerful tool for studying antibody-antigen reactions with a high spatial and temporal resolution.     

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.     

Electron microscopy of subnanometer surface features on metal-decorated protein crystals

Crystals of heavy riboflavin synthase from Bacillus subtilis were freeze-etched and vacuum-coated at normal incidence with 0.1 to 0.4 nm of gold and silver, respectively. This decoration technique was applied to probe the protein surface for preferential nucleation sites. Image processing of the electron micrographs revealed two particular decoration sites for silver and a different one for gold. According to X-ray crystallography, the riboflavin synthase molecules are spherical and smooth except for a surface corrugation of less than 1 nm, which can not be depicted by heavy-metal shadowing. Thus the decoration sites represent sites of specific physical-chemical interactions between the condensing metal and the protein. The decoration pattern correctly reflects the icosahedral symmetry of the almost spherical protein molecules. Owing to the molecule's symmetry, the position of these topochemical sites with respect to the symmetry axes can be localized within 5A. The packing of the molecules in the crystal can be directly observed on shadowed replicas. Only decoration, however, makes it possible to observe the exact orientation of the molecules within the crystal planes and to derive the true lattice constant along the 6-fold screw axis. This proves decoration to be a technique suitable for studying crystal packing and the molecular symmetry of protein complexes at high resolution. The technique can be applied to crystals that are not large enough or insufficiently ordered for X-ray crystallography.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Near-Field Optical Analysis of Plasmonic Nano-Probes for Top-Illumination Tip-Enhanced Raman Scattering

Top-illumination tip-enhanced Raman scattering (TI-TERS) has recently emerged as a promising near-field vibrational spectroscopy method that can be adapted on an upright optical microscope. With a relatively simplified optics, TI-TERS can probe both opaque and transparent samples making them a promising tool in nanoscale chemical analysis. One of the critical components of TI-TERS is the plasmonic nano-tip used to enhance the Raman spectroscopic signature. Herein, we numerically studied the near-field optical properties of conventional gold tip (20 nm radius of curvature) and two varieties of optical antenna-based tips in the context of TI-TERS. Optical antenna-based tips included a 40-nm gold nanoparticle attached to a dielectric tip and a 50-nm equilateral gold nanotriangle attached to a dielectric tip. We evaluated the Raman enhancement spectra as a function of experimental variables such as underlying substrate and angle of the tip with respect to substrate normal. Our analysis revealed that conventional gold tip facilitates superior enhancement and optical antenna-based tips facilitate superior spectral bandwidth and lateral resolution in TI-TERS configuration. Tips with higher enhancement can be harnessed for ultra-sensitive measurements, and tips with broader spectral bandwidth can be utilized to enhance both Stokes and anti-Stokes component of the Raman spectra.     

A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5-15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5–15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

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.     

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA² (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes'' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.     

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.     

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.     

Therapeutic possibilities of plasmonically heated gold nanoparticles

Nanoparticles of gold, which are in the size range 10-100 nm, undergo a plasmon resonance with light. This is a process whereby the electrons of the gold resonate in response to incoming radiation causing them to both absorb and scatter light. This effect can be harnessed to either destroy tissue by local heating or release payload molecules of therapeutic importance. Gold nanoparticles can also be conjugated to biologically active moieties, providing possibilities for targeting to particular tissues. Here, we review the progress made in the exploitation of the plasmon resonance of gold nanoparticles in photo-thermal therapeutic medicine.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Image blurring by thermal diffusion in the observation of hydrated biomolecules with soft X-ray microscopy.

A simple model is presented for the estimation of image blurring in X-ray microscopy of biological specimens in a hydrated environment. The model is essentially based on thermal diffusion of an object to be imaged. The degree of image blurring by diffusion depends on the following situations of the object. The object is free from, is tightly fixed to, or is partially connected to the surrounding structures. The proper imaging time required to achieve a given resolution in X-ray microscopy of biological structures was estimated with the present method. The results suggest that imaging time shorter than 3 msec (free) to 1.4 sec (tightly fixed) is required for the observation of a cell (30 microns in diameter) at the resolution of 100 nm. The model is also applicable to a fragmented object caused by imaging X-rays.     

Investigation of Gold and Silver Nanoparticles on Absorption Heating and Scattering Imaging

Efficient conversion of absorbed light to heat energy and strong scattering by gold and silver nanoparticles suggest these nanoparticles as the agents of heating and imaging. Absorption efficiency and scattering efficiency of gold and silver nanoparticles were studied through numerical simulation using the discrete dipole approximation method. This study shows that the size of gold and silver nanoparticles can effect gold and silver nanoparticles’ absorption efficiency and scattering efficiency. The gold nanoparticle is found to possess the maximum absorption efficiency when the size of gold nanoparticle is 50 nm and the incident wavelength is 540 nm, and the increasing scattering efficiency with the increasing size of gold nanoparticle in the medium, and refractive index of the medium is around 1.33. However, the silver nanoparticle owns the maximum absorption efficiency when the size of silver nanoparticle is 20 nm and the incident wavelength is 396 nm, and the maximum scattering efficiency when the size of silver nanoparticle is 30 nm and the incident wavelength is 410 nm in the same medium. The conditions for achieving the maximum adsorption efficiency and scattering efficiency of gold and silver nanoparticle can be used for heating and imaging using visible and near-infrared light.