Characterization of cellulose whiskers and their nanocomposites by atomic force and electron microscopy

The aim of this work was to compare and explore electron microscopy and atomic force microscopy (AFM) for structure determination of cellulose whiskers and their nanocomposite with poly(lactic acid). From conventional bright-field transmission electron microscopy (TEM) it was possible to identify individual whiskers, which enabled determination of their sizes and shape. AFM overestimated the width of the whiskers due to the tip-broadening effect. Field emission scanning electron microscopy (FESEM) allowed for a quick examination giving an overview of the sample; however, the resolution was considered insufficient for detailed information. Ultramicrotomy of nanocomposite films at cryogenic temperatures enabled detailed inspection of the cellulose whiskers in the poly(lactic acid) matrix by AFM. FESEM applied on fractured surfaces allowed insight into the morphology of the nanocomposite, although rather restricted due to the metal coating and limited resolution. Detailed information was obtained from TEM; however, this technique required staining and suffered in general from limited contrast and beam sensitivity of the material.     

Investigation on the pH‐independent photoluminescence emission from carbon dots impregnated on polymer matrix

Highly luminescent, polymer nanocomposite films based on poly(vinyl alcohol) (PVA), and monodispersed carbon dots (C‐dots) derived from multiwalled carbon nanotubes (MWCNTs), as coatings on substrates as well as free standing ones are obtained via solution‐based techniques. The synthesized films exhibit pH‐independent photoluminescence (PL) emission, which is an advantageous property compared with the pH‐dependent photoluminescence intensity variations, generally observed for the C‐dots dispersed in aqueous solution. The synthesized C‐dots and the nanocomposite films are characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X‐ray diffraction (XRD), Fourier transform infra‐red spectroscopy ( FTIR), ultraviolet (UV) ? visible spectroscopy and photoluminescence spectroscopy (PL) techniques. The TEM image provides clear evidence for the formation of C‐dots of almost uniform shape and average size of about 8 nm, homogeneously dispersed in aqueous medium. The strong anchoring of C‐dots within the polymer matrix can be confirmed from the XRD results. The FTIR spectral studies conclusively establish the presence of oxygen functional groups on the surfaces of the C‐dots. The photoluminescence (PL) emission spectra of the nanocomposite films are broad, covering most part of the visible region. The PL spectra do not show any luminescence intensity variations, when the pH of the medium is changed from 1 to 11. The pH‐independent luminescence, shown by these films offers ample scope for using them as coatings for designing diagnostic and imaging tools in bio medical applications. The non‐toxic nature of these nanocomposite films has been established on the basis of cytotoxicity studies.     

Morphology and primary crystal structure of a silk-like protein polymer synthesized by genetically engineered Escherichia coli bacteria

The morphology and primary crystal structure of SLPF, a protein polymer produced by genetically engineered Escherichia coli bacteria, were characterized. SLPF is a segmented copolymer consisting of amino acid sequence blocks modeled on the crystalline segments of silk fibroin and the cell attachment domain of human fibronectin. Wide angle x-ray scattering (WAXS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and molecular simulations were used to analyze the primary crystal structure of SLPF. TEM experiments conducted on SLPF droplets cast from formic acid on amorphous carbon film demonstrated that these protein films have a microstructure formed of woven sheaves. The sheaves are composed of well-defined whisker crystallites. The width of the whiskers, 11.8 ± 2.2 nm, may be correlated to the length of the silk-like segment in SLPF as predicted by molecular simulations. WAXS data, TEM images, SAED, patterns, molecular simulations, and theoretical diffraction patterns all were consistent with the crankshaft model proposed for Silk I by Lotz and Keith. © 1994 John Wiley & Sons, Inc.     

The Role of Electron Affinity in Determining Whether Fullerenes Catalyze or Inhibit Photooxidation of Polymers for Solar Cells

Understanding the stability and degradation mechanisms of organic solar materials is critically important to achieving long device lifetimes. Here, an investigation of the photodegradation of polymer:fullerene blend films exposed to ambient conditions for a variety of polymer and fullerene derivative combinations is presented. Despite the wide range in polymer stabilities to photodegradation, the rate of irreversible polymer photobleaching in blend films is found to consistently and dramatically increase with decreasing electron affinity of the fullerene derivative. Furthermore, blends containing fullerenes with the smallest electron affinities photobleached at a faster rate than films of the pure polymer. These observations can be explained by a mechanism where both the polymer and fullerene donate photogenerated electrons to diatomic oxygen to form the superoxide radical anion which degrades the polymer.     

UV light‐tunable fluorescent inks and polymer hydrogel films based on carbon nanodots and lanthanide for enhancing anti‐counterfeiting

Novel water‐soluble green fluorescent carbon nanodots (CNs) using methacrylic acid and m‐phenylenediamine as precursors were first synthesized using a one‐pot hydrothermal method. Red fluorescent lanthanide complexes were prepared using lanthanide ion Eu³⁺ and pyridine‐2,6‐dicarboxylic acid. The optical properties of CNs were characterized using ultraviolet visible (UV) spectra and fluorescence spectra, microscopic morphology was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the elemental composition was characterized using Fourier transform‐infrared spectroscopy (FT‐IR) and X‐ray photoelectron spectra (XPS). The fluorescence spectra of the lanthanide complexes were also measured. A simple strategy was developed to prepare UV light‐tunable fluorescent inks and polymer hydrogels films based on CNs and lanthanide complexes. The fluorescent inks and polymer hydrogels films could be repeatedly switched between green and red fluorescence. The change of color depended on luminescence of the CNs and the lanthanide complexes under 254 and 365 nm UV light, respectively. The UV light‐tunable fluorescent inks and polymer hydrogels films could enhance its anti‐counterfeiting function for data and information.     

Orientational order in surface layers of pullulan films

A study was made of spontaneous surface birefringence observed in pullulan films when a tilted polarized beam is passed through a plain polymer film. Birefringence was explored as a function of the angle of beam incidence and the film thickness. The orientational order parameter was estimated for pullulan chains located in the near-surface layers of films. The results were compared with data from earlier studies of other polysaccharides.     

Conversion of the energy of a high-current REB into electromagnetic wave energy

Results are presented from a theoretical investigation and quantitative analysis of the physical processes that govern the efficiency of a coaxial device aimed at converting the energy of a relativistic electron beam into the energy of a TEM wave (a wave in a circular cylindrical coaxial waveguide). The key diffractional problem is solved exactly using a simplified theoretical model, which makes it possible to understand the mechanisms for the formation of a TEM wave and determine how the beam parameters and the design parameters of the converter affect the relative fractions of the kinetic energy of a relativistic electron beam and the energy of its own magnetic and electric fields that are transferred into the energy of the TEM wave field. The results obtained are analyzed quantitatively, and prospects for further theoretical and experimental research in this area are outlined.     

REDUCTION OF HEATING ARTIFACTS IN THIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Certain phenomena affecting contrast obtained from tissue sections with the electron microscope have been investigated and a technique is described for reducing destruction by the electron beam of fine details in sections. It has been concluded that loss of embedding material is slightly higher at exposed surfaces of sections than it is at surfaces covered by substrate film. Covering of both surfaces of sections with thin films of formvar, collodion, or carbon materially improves the general appearance, reduces distortion, and sometimes reduces loss of tissue mass from the section as result of exposure to the electron beam. This improvement is considered to result from the relatively high melting-point of the covering films which serve to eliminate or reduce surface-tension or other forces operating in methacrylate softened by the electron beam.     

Atomic‐Resolution Imaging of Halide Perovskites Using Electron Microscopy

Atomic‐resolution imaging of halide perovskites (HPs) using transmission electron microscopy (TEM) is challenging because of the sensitivity of their structures to the electron beam. In this article, recent achievements in this area are reviewed, covering both all‐inorganic and organic–inorganic hybrid HPs, with an emphasis on the specific imaging conditions that have proven to be effective in avoiding electron beam‐induced structural damage. The discussion focusses on the total electron dose that HPs can bear before being damaged and the effects of different imaging modes, accelerating voltages, and temperatures. The crucial role of a direct‐detection electron‐counting camera in reducing the required electron dose is outlined, which is indispensable for imaging extremely sensitive organic–inorganic hybrid perovskites. In addition to reviewing published works, the results of initial attempts to perform atomic‐resolution elemental mapping for an all‐inorganic HP and image a hybrid HP using scanning TEM are introduced. The preparation of a TEM specimen from macroscopic crystals or devices of HPs, which is very important for practical applications but has not yet received attention, is also discussed. This article aims to provide guidance on the acquisition of atomic‐resolution TEM images of HPs and inspire the development of more imaging technologies for sensitive materials.     

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

Synthesis and characterization of hydroxypropyl methylcellulose and ethyl acrylate graft copolymers

The synthesis of hydroxypropyl methylcellulose-g-poly (ethyl acrylate) was carried out by potassium persulfate induced graft copolymerization in homogeneous aqueous medium. By varying the reaction conditions, graft copolymers with different percentage of grafting were prepared. These graft copolymers were characterized by fourier transform infrared spectra (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analyses (TGA), X-ray diffraction analysis (XRD), and dynamic light scattering (DLS) methods. The molecular weight of grafted and ungrafted polymer chains determined by gel permeation chromatography (GPC) increased with increasing monomer and matrix concentration but decreased with increasing initiator concentration and reaction temperature. The mechanical properties of graft copolymers were measured as function of the percentage of grafting. In addition, the equilibrium humidity adsorption behavior and the disintegration time of the grafted copolymer films were also studied.     

Bacterial cellulose nanocrystals exhibiting high thermal stability and their polymer nanocomposites

Nanocrystals prepared from bacterial cellulose are considered as 'green nanomaterials' depending on their renewable nature and ease of production without the involvement of hazardous chemical treatments. In this investigation, a top down approach was followed for the preparation of bacterial cellulose nanocrystals (BCNC) using a commercially available cellulase enzyme so as to retain native properties of bacterial cellulose even in its nanodimensional form. The morphological and dimensional parameters of BCNC were studied using atomic force microscope (AFM) and transmission electron microscope (TEM). Thermal properties of BCNC produced using the novel enzyme treatment and conventional sulfuric acid hydrolysis were compared. The thermal stability of enzyme processed BCNC was almost two fold higher than sulfuric acid processed ones. Further, the activation energy required for decomposition of enzyme processed BCNC was much higher than the other. Using this enzyme processed BCNC, Polyvinylalcohol (PVA) nanocomposite films were prepared and characterized. Incorporation of these nanocrystals in polymer matrix resulted in a remarkable improvement in the thermal stability as well as mechanical properties of nanocomposite films. These nanocomposites exhibited higher melting temperature (Tm) and enthalpy of melting (ΔHm) than those of pure PVA, suggesting that the addition of nanocrystals modified the thermal properties of PVA. The effective load transfer from polymer chains to the BCNC resulted in an improved tensile strength from 62.5 MPa to 128 MPa, by the addition of just 4 wt% of BCNC. Furthermore, the elastic modulus was found to increase from 2 GPa to 3.4 GPa. The BCNC obtained through cellulose treatment under controlled conditions were associated with several desirable properties and appear to be superior over the conventional methods of nanocrystals production. The enzymatic method followed in this study is expected to contribute the fabrication of high performance polymer nanocomposites in a much greener and innovative manner.     

In vivo EBT radiochromic film dosimetry of electron beam for Total Skin Electron Therapy (TSET)

EBT radiochromic films were used to determine skin-dose maps for patients undergone Total Skin Electron Therapy (TSET). Gafchromic EBT radiochromic film is one of the newest radiation-induced auto-developing photon and electron-beam analysis films available for therapeutic radiation dosimetry in radiotherapy applications. EBT films can be particularly useful in TSET; due to patient morphology, underdosed regions typically occur, and the radiochromic film represents a suitable candidate for monitoring them.In this study, TSET was applied to treat cutaneous T-cell lymphoma. The technique for TSET was implemented by using an electron beam with a nominal energy of 6 MeV. The patient was treated in a standing position using dual angled fields in order to obtain the greatest dose uniformity along the patient's longitudinal axis. The electron beam energy was degraded by a PMMA filter. The in vivo dose distribution was determined through the use of EBT films, as well as of thermoluminescent dosimeters for comparison (TLDs). EBT results showed a reasonable agreement with TLDs data.     

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.     

Core-shell Au nanoparticle formation with DNA-polymer hybrid coatings using aqueous ATRP

We report here a direct surface-grafting approach to forming DNA-containing polymer shells outside of Au nanoparticles using aqueous atom transfer radical polymerization (ATRP). In this approach, DNA molecules were immobilized on Au particles to introduce ATRP initiators on the surface. The same DNA molecules also acted as particle stabilizers through electrostatic repulsion and allowed particles to stay suspended in water. The immobilized ATRP initiators prompted polymer chain growth under certain conditions to form thick polymer shells outside of the particles. The formation of DNA-polymer hybrids outside of Au nanoparticles was characterized using absorption spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and gel electrophoresis. The presence of thick polymer shells improved particle stability in high ionic strength media, whereas particles with the DNA coating only aggregated. A visible color difference between these two particle solutions was clearly observed, providing the basis for DNA sensing in homogeneous solutions.     

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.     

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.     

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.     

Applications of field emission scanning electron microscopy to polymer membrane research

The recent development of ultra-high resolution field emission scanning electron microscopy has opened exciting new opportunities in many scientific and engineering applications at the molecular scale. It overcomes the instrumentation limitations of low resolution in SEM and uncertainty in TEM due to artifacts imposed by sample preparation.Applications of field emission scanning electron microscopy (FESEM) to polymer membrane research such as studies of surface morphology of finely porous membranes and mechanisms of membrane fouling are illustrated with examples. The advantages of the technique, especially the low voltage requirements of FESEM for surface observation, are also discussed in comparison with TEM (replica) and conventional SEM.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.