Endocytosis in Trypanosoma cruzi (Euglenozoa: Kinetoplastea) epimastigotes: Visualization of ingested transferrin-gold nanoparticle complexes by confocal laser microscopy

Here we describe the visualization by confocal microscopy of ingested gold (15nm)-labeled transferrin in epimastigote forms of the protozoan Trypanosoma cruzi. Intracellular gold labeling was evident at two sites, which represent the bottom of the cytopharynx and the reservosomes. The gold tracer was best observed by confocal microscopy by using the 633nm excitation wavelength. Intracellular gold clusters larger than 60nm could be visualized by either gold reflection (light scattering) or photoluminescence modes. The gold reflection mode, the gold photoluminescence mode and the anti-transferrin immunofluorescence image of gold-labeled transferrin showed co-localization, thus demonstrating that the gold visualization modes did not represent artifacts or mislocalization of the biomarker. Visualization of protein-gold nanoparticle complexes by confocal microscopy thus emerges as a promising imaging tool to explore the endocytic pathway in trypanosomes and other cell types, as well as to perform immunolocalization studies using gold-labeled secondary antibodies.     

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.     

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.     

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

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.     

Liquid phase SPR imaging experiments for biosensors applications

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

FluoroNanogold: an important probe for correlative microscopy

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.     

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.     

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.     

Colloidal gold particles as an incompressible atomic force microscope imaging standard for assessing the compressibility of biomolecules.

Colloidal gold particles have multiple uses as three-dimensional atomic force microscopy imaging standards because they are incompressible, monodisperse, and spherical. The spherical nature of the particles can be exploited to characterize scanning tip geometry. As uniform spheres, colloidal gold particles may be used to calibrate the vertical dimensions of atomic force microscopy at the nanometer level. The monodisperse and incompressible nature of the gold can be used to characterize the vertical dimensions of coadsorbed biomolecules. Simultaneous measurements of gold with tobacco mosaic virus show that, at the same applied vertical force, the tobacco mosaic virus is undamaged by blunt tips but is compressed or disintegrated under sharper scanning styli, suggesting that specimen degradation is partly a pressure-dependent effect.     

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.     

Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy

Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.     

ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.     

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.     

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.     

Designable nanoplasmonic biomarkers for direct microscopy cytopathology diagnostics

Direct microscopy interpretation of fine‐needle biopsy cytological samples is routinely used by practicing cytopathologists. Adding possibility to identify selective and multiplexed biomarkers on the same samples and with the same microscopy technique can greatly improve diagnostic accuracy. In this article, we propose to use biomarkers based on designable plasmonic nanoparticles (NPs) with unique optical properties and excellent chemical stability that can satisfy the above‐mentioned requirements. By finely controlling the size and composition of gold‐silver alloy NPs and gold nanorods, the NPs plasmonic resonance properties, such as scattering efficiency and resonance peak spectral position, are adjusted in order to provide reliable identification and chromatic differentiation by conventional direct microscopy. Efficient darkfield NPs imaging is performed by using a novel circular side illumination adaptor that can be easily integrated into any microscopy setup while preserving standard cytopathology visualization method. The efficiency of the proposed technology for fast visual detection and differentiation of three spectrally distinct NP‐markers is demonstrated in different working media, thus confirming the potential application in conventional cytology preparations. It is worth emphasizing that the presented technology does not interfere with standard visualization with immunohistochemical staining, but should rather be considered as a second imaging modality to confirm the diagnostics.      

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy.

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.     

Evaluation of Oolong Tea Extract Staining of Brain Tissue with Special Reference to Smooth Endoplasmic Reticulum

Biophysics - Electron microscopy remains the gold standard for studying the nervous system, as it allows adequate spatial resolution imaging of the finer structures of nervous tissues. Despite the...