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.     

Methods of detection and identification of manufactured nanoparticles

The actual methods of detection and identification of manufactured nanoparticles in both simple and complex multi-component matrix for assessing biological effects and safety of nanotechnology products have been reviewed. The detection of priority species of biologically active nanoparticles, which include fullerenes, singleand multi-walled carbon nanotubes, nanoparticles of silver, gold, titanium oxide, aluminum, cerium, zinc and silicon, has been given a special attention. The requirements for sample preparation have been discussed. The results of the successful application for the detection of manufactured nanoparticles in biosamples with methods of scanning and transmission electron microscopy, confocal laser scanning microscopy, atomic force microscopy, scanning tunneling microscopy, size exclusion chromatography, field-flow fractionation, electrophoretic, light scattering, spectrophotometry, fluorescent spectroscopy, X-ray and other spectrometry, mass spectrometry, “particle counters”, immunochemistry have been reviewed. The possibilities and limitations of different techniques, and their complementarity have been analyzed.     

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of > 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.     

Synthesis and characterization of zinc oxide nanoparticles by using polyol chemistry for their antimicrobial and antibiofilm activity

The present investigation deals with facile polyol mediated synthesis and characterization of ZnO nanoparticles and their antimicrobial activities against pathogenic microorganisms. The synthesis process was carried out by refluxing zinc acetate precursor in diethylene glycol(DEG) and triethylene glycol(TEG) in the presence and in the absence of sodium acetate for 2 h and 3 h. All synthesized ZnO nanoparticles were characterized by X-ray diffraction (XRD), UV visible spectroscopy (UV), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy(FESEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX) technique. All nanoparticles showed different degree of antibacterial and antibiofilm activity against Gram-positive Staphylococcus aureus (NCIM 2654)and Gram-negative Proteus vulgaris (NCIM 2613). The antibacterial and antibiofilm activity was inversely proportional to the size of the synthesized ZnO nanoparticles. Among all prepared particles, ZnO nanoparticles with least size (~ 15 nm) prepared by refluxing zinc acetate dihydrate in diethylene glycol for 3 h exhibited remarkable antibacterial and antibiofilm activity which may serve as potential alternatives in biomedical application.     

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.     

Biosynthesis, characterization and antibacterial activity of silver nanoparticles by aqueous Annona squamosa L. leaf extract at room temperature

Silver nanoparticles (AgNPs) have gained great interest in nanotechnology, biotechnology and medicine. The green synthesis of nanoparticles has received an increasing attention because of it’s maximize efficiency and minimize health and environmental hazards as compared to other conventional chemical synthesis. In this study, we reported biosynthesis of AgNPs by aqueous Annona squamosa L. leaf extract and its characterization by UV-visible spectroscopy (UV–vis), Field emission gun scanning electron microscopy (FEG-SEM), X-ray energy dispersive spectroscopy (EDX), Transmission electron microscopy (TEM), Selected-area electron diffraction (SAED) and Fourier transform infra-red spectroscopy (FTIR). The results indicated that AgNPs formed were spherical in shape with size ranging from 14 to 40 nm with an average diameter 28.47 nm. Furthermore, it was observed that the AgNPs exhibited an antibacterial activity against different Gram positive and Gram negative microorganisms. Our report confirmed that the ALE is a very good eco-friendly and nontoxic bioreductant for the synthesis of AgNPs and opens up further opportunities for fabrication of antibacterial drugs, medical devices and wound dressings.     

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R² = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.     

Microscopies,spectroscopies and spectrometries applied to marine corrosion of copper

Used in combination, surface analytical techniques can resolve spatial relationships between bacteria and localized corrosion, determine specific corrosion mechanisms and differentiate between abiotic and biotic processes. Confocal laser scanning microscopy and scanning vibrating electrode microscopy were used to demonstrate that marine bacteria and anodic sites are co‐located. Environmental scanning electron microscopy coupled with energy dispersive X‐ray spectros‐copy was used to demonstrate dealloying of nickel from copper: nickel alloys. X‐ray absorption spectroscopy, and transmission electron microscopy equipped with electron energy loss spectrometry were used to determine the speciation of copper associated with corrosion products.     

Elucidating the chemical and biochemical applications of Citrus sinensis-mediated silver nanocrystal

Abstract

Synthesis of nanoparticles using biodegradable source is safer and echo-friendly. Here, we describe the synthesis of polycrystalline silver nanocrystals using Citrus sinensis acting as both reducing and capping agents. After exposing the silver ions to orange extract, rapid reduction of silver ions led to the formation of stable silver nanocrystals due to the reducing and stabilizing properties of orange fruit juice. The synthesized silver nanocrystals were characterized using various analytical techniques like UV–vis spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscopy (TEM). The biochemical activity of the synthesized nanocrystals was studied in the light of affinity to bovine serum albumin using several biophysical methods like absorbance, fluorescence and circular dichroism spectroscopy. Cytotoxic activity of these nanocrystals was also studied against Hep-2 cell line using fluorescence microscopy. It was also found that the synthesized nanocrystals can sense mercuric ion down to 50?µM in the presence of a number of cations. Furthermore, we established that the silver nanoparticles can effectively catalyse the reduction of methylene blue by ascorbic acid. The present study will enrich our knowledge on the chemical and biochemical activities of green-synthesized silver nanocrystals.     

Novel polyhedral gold nanoparticles: green synthesis,optimization and characterization by environmental isolate of Acinetobacter sp. SW30

Gold nanoparticles have enormous applications in cancer treatment, drug delivery and nanobiosensor due to their biocompatibility. Biological route of synthesis of metal nanoparticles are cost effective and eco-friendly. Acinetobacter sp. SW 30 isolated from activated sewage sludge produced cell bound as well as intracellular gold nanoparticles when challenged with HAuCl₄ salt solution. We first time report the optimization of various physiological parameters such as age of culture, cell density and physicochemical parameters viz HAuCl₄ concentration, temperature and pH which influence the synthesis of gold nanoparticles. Gold nanoparticles thus produced were characterized by various analytical techniques viz. UV–Visible spectroscopy, X-ray diffraction, cyclic voltammetry, transmission electron microscopy, selected area electron diffraction, high resolution transmission electron microscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy and dynamic light scattering. Polyhedral gold nanoparticles of size 20 ± 10 nm were synthesized by 24 h grown culture of cell density 2.4 × 10⁹ cfu/ml at 50 °C and pH 9 in 0.5 mM HAuCl₄. It was found that most of the gold nanoparticles were released into solution from bacterial cell surface of Acinetobacter sp. at pH 9 and 50 °C.     

Structural and optical properties of dysprosium-doped hydroxyapatite nanoparticles and the use as a bioimaging probe in human cells

In this work, the hydroxyapatite nanoparticles doped with trivalent dysprosium ions were synthesized by a co-precipitation method. The characterization techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) were carried out to determine the crystalline and structural properties. The Rietveld structural refinement of the XRD patterns confirmed the purity of the phase formation of the synthesized nanoparticles. The photoluminescence emission spectra exhibited intense emissions in the blue region at 450 nm and 476 nm along with less intense yellow emission at 573 nm which can be attributed to the magnetic dipole and electric dipole transitions of dysprosium respectively. In order to evaluate the colour tunability of the emitted light CIE chromaticity coordinate values were calculated. The intense blue emissions from the synthesized sample were found to be favourable for bioimaging. The images obtained from the fluorescence microscopy revealed that the dysprosium-doped hydroxyapatite nanoparticles are potential bioimaging probes in human cells.     

Binding interaction study on human serum albumin with bactericidal gold nanoparticles synthesized from a leaf extract of Musa balbisiana: a multispectroscopic approach

This study describes the eco‐friendly, low‐cost and room‐temperature synthesis of gold nanoparticles from Musa balbisiana leaf extract, which acts as both reducing and stabilizing agent, and characterized by ultraviolet?visible (UV–vis) light spectroscopy, fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FE‐SEM), analytical transmission electron microscopy (TEM), energy‐dispersive X‐ray spectroscopy (EDAX) and dynamic light scattering (DLS) instruments. These nanoparticles showed an average diameter of 33.83 ± 3.39 nm, which was confirmed from the size distribution histogram. The bactericidal activity of these nanoparticles was confirmed using bacteria Escherichia coli and Staphylococcus aureus at 1 and 2 nM minimum inhibitory concentrations, respectively. The interaction between nanoparticles and human serum albumin (HSA) was investigated, as this plays significant roles in biological systems. The nature of interaction, binding parameters and structural variation of HSA in the presence of these nanoparticles have been evaluated using several useful spectroscopic approaches such as UV–vis, FTIR, time‐resolved and steady‐state fluorescence, and circular dichroism in addition to the measurement of zeta potential. This interaction study revealed that static quenching occurs in this process with minimal alteration in the secondary structure, but the native structure of HSA remained unaltered. The binding constant and thermodynamic parameters of this interaction process were also evaluated.     

Biofabrication of platinum nanoparticles using Fumariae herba extract and their catalytic properties

Due to the increasing popularity of using plant extract in the synthesis of nanoparticles, this study presented the synthesis of platinum nanoparticles using Fumariae herba extract. The formation of platinum nanoparticles was confirmed by UV–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) with EDS profile. Transmission electron micrograph presented the hexagonal and pentagonal shape of the synthesized nanoparticles sized about 30 nm. Moreover, platinum nanoparticles presented good catalytic properties in the reduction of methylene blue and crystal violet.     

An intracellular silver deposition method for targeted detection and chemical analysis of uncultured microorganisms

The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (¹³C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

The blood-brain barrier penetration and distribution of PEGylated fluorescein-doped magnetic silica nanoparticles in rat brain

PEGylated PAMAM conjugated fluorescein-doped magnetic silica nanoparticles (PEGylated PFMSNs) have been synthesized for evaluating their ability across the blood-brain barrier (BBB) and distribution in rat brain. The obtained nanoparticles were characterized by transmission electron microscopy (TEM), thermal gravimetry analyses (TGA), zeta potential (ζ-potential) titration, and X-ray photoelectron spectroscopy (XPS). The BBB penetration and distribution of PEGylated PFMSNs and FMSNs in rat brain were investigated not only at the cellular level with Confocal laser scanning microscopy (CLSM), but also at the subcellular level with transmission electron microscopy (TEM). The results provide direct evidents that PEGylated PFMSNs could penetrate the BBB and spread into the brain parenchyma.     

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.     

Luminescence investigation of a cerium‐doped yttrium vanadate phosphor

Cerium (Ce³⁺)‐doped (1, 3, and 7 mol%) yttrium vanadate phosphors were prepared using a co‐precipitation technique. The structural and optical properties of the synthesized samples were studied using X‐ray diffraction (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), high‐resolution transmission electron microscopy (HR‐TEM), optical absorption, and photoluminescence (PL) spectroscopy techniques. The tetragonal structure and the formation of the nanosized crystallites in the YVO₄:Ce phosphor were confirmed using XRD analysis. HR‐TEM morphology showed rod‐like nanoparticles of different sizes. Optical absorption spectra demonstrated strong absorption bands at 268 and 276 nm. PL spectra showed strong peaks at 546, 574, and 691 nm following excitation at 300 nm. The calculated CIE chromaticity coordinates demonstrated that YVO₄:Ce could be used as a novel phosphor for the development of light‐emitting diode lamps.     

Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules

Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.     

Microwave-assisted synthesis of praseodymium (Pr)-doped ZnS QDs such as nanoparticles for optoelectronic applications

Praseodymium (Pr)-doped ZnS nanoparticles were synthesized using a low-cost microwave-assisted technique and investigations on their structure, morphology, optical properties, Raman resonance, dielectric properties, and luminescence were conducted. Broad X-ray diffraction peaks suggested the formation of low-dimensional Pr-doped ZnS nanoparticles with a cubic structure that was validated using transmission electron microscopy (TEM)/high-resolution TEM analysis. The energy gaps were identified using diffuse reflectance spectroscopy and it was found that the values varied between 3.54eV and 3.61eV for different samples. Vibrational experiments on Pr-doped ZnS nanoparticles revealed significant Raman modes at ~270 and ~350 cm⁻¹ that were associated with optical phonon modes that are shifted to lower wavenumbers, indicating phonon confinement in the synthesized products. The photoluminescence (PL) spectra of all samples demonstrated that the pure and Pr-doped ZnS nanoparticles were three-level laser active materials. Energy-dispersive X-ray spectroscopy and mapping study confirmed the homogeneous presence of Pr in ZnS. TEM studies showed that the particles were of very small size and in the cubic phase. The samples had high dielectric constant values between 13 and 24 and low loss values, according to the dielectric analysis. With an increase in frequency and a change in the Pr content of ZnS, an intense peak could be seen in the PL spectra at a wavelength of 360 nm, and some other peaks observed corresponded to the transition of Pr³⁺. The produced nanoparticles were appropriate for optoelectronic applications due to their short dimension, high energy gap, high dielectric constant, and low loss values.