Transmission-mode Microscope Research Articles

Traceable determination of the size of nanoparticles up to 500 nm by scanning electron microscopy in transmission mode based on simulation

The size measurement of nanoparticles contributes to the understanding of their properties and, thus, to the assessment of the risks they pose to health and the environment. For such measurements to be comparable and legally recognized, they must be traceable to the SI unit meter. Recently, interest in traceable measurements of polystyrene particles with sizes up to 500 nm has aroused, e.g. in the aerosol community. To meet this demand, we adapted an established method to traceably measure nanoparticles with high precision in the transmission mode of a scanning electron microscope (STEM-in-SEM or TSEM). Since this method was geared towards smaller particles, we adapted it at two points: New simulations with the program Geant4SEM allow a more accurate modelling, especially of the inelastical scattering processes. In addition, the image evaluation procedure was revised to account for the non-linear signal response at the particle boundary. The measured values obtained in this manner show good agreement with the values of two international intercomparisons.

Influence of Surfactant Concentration on Spontaneous Emulsification Kinetics

The kinetics of spontaneous emulsification is investigated on aqueous pendant drops in paraffin oil. Optical microscopy in transmission mode is used for high-spatial-resolution image recording. The influence of a lipophilic surfactant (Span 80) and two water-soluble surfactants (CTAB and SDS) is investigated. As time runs, the drop interface turns opaque due to the formation of microstructures associated with spontaneous emulsification. The time evolution of this phenomenon is shown to depend upon temperature and surfactant concentration, which leads to an overall shrinkage due to gradual water uptake and transport into paraffin oil. Spontaneous emulsification kinetics depends upon the chemical composition. Higher concentrations of Span 80 and CTAB (resp. SDS) are shown to promote (resp. hinder) water transport. This work provides new insights into the understanding of spontaneous emulsification when combining the properties of non-ionic and ionic surfactants.

Nucleation and growth kinetics of ZnAl[formula omitted]O[formula omitted] spinel in crystalline ZnO – amorphous Al[formula omitted]O[formula omitted] bilayers prepared by atomic layer deposition

Applying scanning electron microscopy in transmission mode and grazing incidence X-ray diffraction, we investigated the spinel-forming solid-state reaction between crystalline ZnO and amorphous Al2O3 layers prepared by atomic layer deposition. We observed two-stage phase growth of the crystalline ZnAl2O4 product layer. During the first stage, flat, pancake-like islands are formed in the nucleation process and these nuclei grow laterally without much increment in thickness. After these islands grow together to form a continuous layer, planar growth is happening in the second stage. We show that the solid-state reaction is governed by grain boundary and interface diffusion instead of interface reaction control observed between crystalline parent phases.

Development of Thermo- and pH-Sensitive Liposomal Magnetic Carriers for New Potential Antitumor Thienopyridine Derivatives.

The development of stimuli-sensitive drug delivery systems is a very attractive area of current research in cancer therapy. The deep knowledge on the microenvironment of tumors has supported the progress of nanosystems’ ability for controlled and local fusion as well as drug release. Temperature and pH are two of the most promising triggers in the development of sensitive formulations to improve the efficacy of anticancer agents. Herein, magnetic liposomes with fusogenic sensitivity to pH and temperature were developed aiming at dual cancer therapy (by chemotherapy and magnetic hyperthermia). Magnetic nanoparticles of mixed calcium/manganese ferrite were synthesized by co-precipitation with citrate and by sol–gel method, and characterized by X-ray diffraction (XRD), scanning electron microscopy in transmission mode (STEM), and superconducting quantum interference device (SQUID). The citrate-stabilized nanoparticles showed a small-sized population (around 8 nm, determined by XRD) and suitable magnetic properties, with a low coercivity and high saturation magnetization (~54 emu/g). The nanoparticles were incorporated into liposomes of dipalmitoylphosphatidylcholine/cholesteryl hemisuccinate (DPPC:CHEMS) and of the same components with a PEGylated lipid (DPPC:CHEMS:DSPE-PEG), resulting in magnetoliposomes with sizes around 100 nm. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements were performed to investigate the pH-sensitivity of the magnetoliposomes’ fusogenic ability. Two new antitumor thienopyridine derivatives were efficiently encapsulated in the magnetic liposomes and the drug delivery capability of the loaded nanosystems was evaluated, under different pH and temperature conditions.

Three-dimensional transfer function of optical microscopes in reflection mode.

Three-dimensional (3D) transfer functions build the basis for a comprehensive characterization of optical imaging systems in the spatial frequency domain. Utilizing the projection-slice theorem, the 2D modulation transfer function of an incoherent imaging system can be derived from a 3D transfer function by integration with respect to the axial spatial frequency. For a diffraction limited microscope with homogeneous incoherent pupil illumination, the modulation transfer function equals the 2D autocorrelation function of a circular disc. However, until now to the best of our knowledge no 3D transfer function has been published, which exactly leads to the 2D modulation transfer function of a diffraction limited microscope in reflection mode. In this article, we derive a formula, which after integration with respect to the axial spatial frequency coordinate perfectly fits to the diffraction limited 2D modulation transfer function. The inverse three-dimensional Fourier transform of the 3D transfer function results in a complex-valued 3D point spread function, from which the depth of field, the lateral resolution and, in addition, the corresponding 3D point spread function of both, a conventional and an interference microscope, can beobtained.

Carrier Fibers for the Safe Dosage of Nanoparticles in Nanocomposites: Nanomechanical and Thermomechanical Study on Polycarbonate/Boehmite Electrospun Fibers Embedded in Epoxy Resin.

The reinforcing effect of boehmite nanoparticles (BNP) in epoxy resins for fiber composite lightweight construction is related to the formation of a soft but bound interphase between filler and polymer. The interphase is able to dissipate crack propagation energy and consequently increases the fracture toughness of the epoxy resin. Usually, the nanoparticles are dispersed in the resin and then mixed with the hardener to form an applicable mixture to impregnate the fibers. If one wishes to locally increase the fracture toughness at particularly stressed positions of the fiber-reinforced polymer composites (FRPC), this could be done by spraying nanoparticles from a suspension. However, this would entail high costs for removing the nanoparticles from the ambient air. We propose that a fiber fleece containing bound nanoparticles be inserted at exposed locations. For the present proof-of-concept study, an electrospun polycarbonate nonwoven and taurine modified BNP are proposed. After fabrication of suitable PC/EP/BNP composites, the thermomechanical properties were tested by dynamic mechanical analysis (DMA). Comparatively, the local nanomechanical properties such as stiffness and elastic modulus were determined by atomic force microscopy (AFM). An additional investigation of the distribution of the nanoparticles in the epoxy matrix, which is a prerequisite for an effective nanocomposite, is carried out by scanning electron microscopy in transmission mode (TSEM). From the results it can be concluded that the concept of carrier fibers for nanoparticles is viable.

Quantitative Polarization-Resolved Second-Harmonic-Generation Microscopy of Glycine Microneedles.

Second-harmonic generation (SHG) is a nonlinear optical process that can provide disease diagnosis through characterization of biological building blocks such as amino acids, peptides, and proteins. The second-order nonlinear susceptibility tensor χ(2) of a material characterizes its tendency to cause SHG. Here, a method for finding the χ(2) elements from polarization-resolved SHG microscopy in transmission mode is presented. The quantitative framework and analytical approach that corrects for micrometer-scale morphology and birefringence enable the determination and comparison of the SHG susceptibility tensors of β- and γ-phase glycine microneedles. The maximum nonlinear susceptibility coefficients are d33 = 15 pm V-1 for the β and d33 = 5.9 pm V-1 for the γ phase. The results demonstrate glycine as a useful biocompatible nonlinear material. This combination of the analytical model and polarization-resolved SHG transmission microscopy is broadly applicable for quantitative SHG material characterization and diagnostic imaging.

Morphology of the mandibular gnathobases of the copepods Calanus australis and Calanoides carinatus: Evidence of omnivory

In spite of the worldwide ecological relevance of Calanus australis and Calanoides carinatus, little is known on the morphology of their mandibular gnathobase edges (MGEs). Thus, in order to further learn about the relationship between MGEs and diet, adults and copepodites of C. australis and C. carinatus collected from northern Patagonian coasts, Argentina, were morphologically analyzed using confocal laser scanning microscopy in transmission mode and scanning electron microscopy. In C. australis males as well as in C. australis and C. carinatus females, MGEs were found to end in one dorsal seta and to have one ventral tooth, four central teeth and three dorsal teeth. In contrast, in C. carinatus males, MGEs were observed to be rudimentary and with no dorsal seta, and −contrary to published data− they were found to have three cone-shaped, short and small tooth-like structures and three-four tooth-like structures that were long and pointed. The main differences between MGEs from copepodites and adults of C. australis and C. carinatus females lied in the number of cuspids observed on the different teeth and the presence of a short inner lateral peak found only in C. australis ventral tooth. Overall, the evidence gathered from our study leads us to classify C. australis and C. carinatus as omnivores.

Structural organization of the cell wall polymers in compression wood as revealed by FTIR microspectroscopy.

Glucomannan was more strongly oriented, in line with the orientation of cellulose, than the xylan in both compression wood and normal wood of Chinese fir. Lignin in compression wood was somewhat more oriented in the direction of the cellulose microfibrils than in normal wood. The structural organization in compression wood (CW) is quite different from that in normal wood (NW). To shed more light on the structural organization of the polymers in plant cell walls, Fourier Transform Infrared (FTIR) microscopy in transmission mode has been used to compare the S2-dominated mean orientation of wood polymers in CW with that in NW from Chinese fir (Cunninghamia lanceolata). Polarized FTIR measurements revealed that in both CW and NW samples, glucomannan and xylan showed a parallel orientation with respect to the cellulose microfibrils. In both wood samples, the glucomannan showed a much greater degree of orientation than the xylan, indicating that the glucomannan has established a stronger interaction with cellulose than xylan. For the lignin, the absorption peak also indicated an orientation along the direction of the cellulose microfibrils, but this orientation was more pronounced in CW than in NW, indicating that the lignin is affected by the orientation of the cellulose microfibrils more strongly in CW than it is in NW.

Microsphere-assisted self-referencing digital holographic microscopy in transmission mode

In this paper, we introduce a self-referencing microsphere-assisted transmission digital holographic microscopy (DHM) system in real-image operating mode. Self-referencing DHM arrangements provide a compact geometry that are temporally stable against environmental vibrations and suitable for the measurement of dynamic specimens such as cells. These advantages may be more pronounced for microsphere-assisted DHM systems. In the real-image mode, unlike the virtual-image mode, the working distance is increased. This, in turn, provides flexibility for insertion of additional elements for enhancement of the image quality, or for other required tasks. The lateral resolution enhancement is similar to the virtual image arrangement and the axial resolution is decoupled from the lateral one. The methodology is discussed theoretically and validated experimentally by conducting DHM experiments on standard micro-objects and aggregation of micro-particles. Our results show that, by the assistance of a rather big size 550 μm silica microsphere, a 10× microscope objective can resolve the 3D structure of a compact disk. The arrangement may be useful toward having a compact and inexpensive bench-top high-resolution three-dimensional imaging apparatus.

Dislocation dynamics in a nickel-based superalloy via in-situ transmission scanning electron microscopy

Micro-tensile specimens of nickel-based superalloy oligocrystals were tested in-situ in an scanning electron microscope in transmission mode (TSEM) enabling observation of dislocations. The dynamics of dislocation motion during tensile loading were captured and correlated with the measured intermittencies during plastic flow recorded by high load- and temporal-resolution sensors. This investigation in particular focused on the dislocation behavior near twin boundaries with different slip configurations. A multiplicity of deformation mechanisms at the dislocation scale were observed within individual slip bands, including precipitate shearing, dislocation decorrelation and antiphase boundary-coupled shearing. These processes affect strain localization near twin boundaries and provide new defect-level insights on plastic localization and fatigue crack initiation in these alloys.

Electron energy-loss spectroscopy (EELS) with a cold-field emission scanning electron microscope at low accelerating voltage in transmission mode

A commercial electron energy-loss spectrometer (EELS) attached to a high-resolution cold-field emission scanning electron microscope in transmission mode (STEM) is evaluated and its potential for characterizing materials science thin specimens at low accelerating voltage is reviewed. Despite the increased beam radiation damage at SEM voltages on sensitive compounds, we describe some potential applications which benefit from lowering the primary electrons voltage on less-sensitive specimens. We report bandgap measurements on several dielectrics which were facilitated by the lack of Cherenkov radiation losses at 30 kV. The possibility of volume plasmon imaging to probe local composition changes in complex materials was demonstrated using energy-filtered STEM, either via spectrum imaging or elemental mapping using the “three-windows” method. As plasmonic materials are increasing used for energy, electronics or biomedical applications, the ability of reliably evaluate their properties at low accelerating voltage in a SEM is very appealing and is demonstrated. The energy resolution of the spectrometer, taken as the full width at half maximum of the zero-loss peak, was routinely measured at around 0.55 eV and it is demonstrated that t/λ ratios up to 1.5 allowed practical EEL spectroscopy at 30 kV.

Low Voltage Analytical Possibilities in a Scanning Electron Microscope in Transmission Mode at 30 kV: EDS, EELS and CBED at the Nanoscale

Low Voltage Analytical Possibilities in a Scanning Electron Microscope in Transmission Mode at 30 kV: EDS, EELS and CBED at the Nanoscale

Fatigue behavior of an ultrafine-grained metastable CrMnNi steel tested under total strain control

An ultrafine-grained (UFG) microstructure in a metastable austenitic CrMnNi steel was achieved using a thermo-mechanically controlled process by rotary swaging and subsequent reversion annealing. The material with an average grain size of 0.7μm was cyclically deformed in total strain controlled tests at strain amplitudes in the range of 0.3%≤Δεt/2≤1.2%. This treatment increased the cyclic stress amplitudes as well as the fatigue life in comparison with the conventionally grained counterpart. For strain amplitudes Δεt/2≥0.4% a martensitic phase transformation occurred, which was observed in situ by a ferrite sensor as an increase of the α′-martensite fraction. The microstructure changes, and the deformation mechanisms in particular, were investigated by means of electron backscatter diffraction, scanning electron microscopy in transmission mode and transmission electron microscopy that revealed the formation of small α′-nuclei which rapidly grew until the entire austenitic grain was transformed.

Study of the Effect of a Polymeric Compatibilizing Agent on the Flame Retardancy and Tensile Properties of High Density Polyethylene/Magnesium Hydroxide Compositions

The effect of using a compatibilizer, such as maleic anhydride grafted polyethylene (PEgMA), on the flame retardancy and mechanical (tensile) properties of HDPE nanocomposites with silane modified Mg(OH)2 was studied. The silane modification of Mg(OH)2 was confirmed by FTIR. Filler dispersion was characterized by scanning electron microscopy in transmission mode (STEM) and the effect of filler on mechanical properties was also evaluated. The flame retardant properties were evaluated by flammability tests (Underwriters Laboratory – UL‐94) and cone calorimeter. The mechanism by which the use of PEgMA compatibilizer improves the flame retardancy of HDPE/Mg(OH)2 was studied. Through the simultaneous TGAIR analysis, it was found that when using PEgMA, the flame retardant properties of HDPE/Mg(OH)2 are modified, due to the generation of volatile compounds in the thermal decomposition.

Non-Diffractive Electron Bessel Beams for Scanning Electron Microscopy in Transmission Mode Using Direct Phase Masks

Non-Diffractive Electron Bessel Beams for Scanning Electron Microscopy in Transmission Mode Using Direct Phase Masks

The qualitative f-ratio method applied to electron channelling-induced x-ray imaging with an annular silicon drift detector in a scanning electron microscope in the transmission mode

Electron channelling is known to affect the x-ray production when an accelerated electron beam is applied to a crystalline material and is highly dependent on the local crystal orientation. This effect, unless very long counting time are used, is barely noticeable on x-ray energy spectra recorded with conventional silicon drift detectors (SDD) located at a small elevation angle. However, the very high count rates provided by the new commercially available annular SDDs permit now to observe this effect routinely and may, in some circumstances, hide the true elemental x-ray variations due to the local true specimen composition. To circumvent this issue, the recently developed f-ratio method was applied to display qualitatively the true net intensity x-ray variations in a thin specimen of a Ti-6Al-4V alloy in a scanning electron microscope in transmission mode. The diffraction contrast observed in the x-ray images was successfully cancelled through the use of f-ratios and the true composition variations at the grain boundaries could be observed in relation to the dislocation alignment prior to the β-phase nucleation. The qualitative effectiveness in removing channelling effects demonstrated in this work makes the f-ratio, in its quantitative form, a possible alternative to the ZAF method in channelling conditions.

CO2 laser polishing of microfluidic channels fabricated by femtosecond laser assisted carving

In this study, we investigate the effects of CO2 laser polishing on microscopic structures fabricated by femtosecond laser assisted carving (FLAC). FLAC is the peripheral laser irradiation of 2.5D structures suitable for low repetition rate lasers and is first used to define the microwell structures in fused silica followed by chemical etching. Subsequently, the bottom surface of patterned microwells is irradiated with a pulsed CO2 laser. The surfaces were characterized using an atomic force microscope (AFM) and scanning electron microscope (SEM) in terms of roughness and high quality optical imaging before and after the CO2 laser treatment. The AFM measurements show that the surface roughness improves more than threefold after CO2 laser polishing, which promises good channel quality for applications that require optical imaging. In order to demonstrate the ability of this method to produce low surface roughness systems, we have fabricated a microfluidic channel. The channel is filled with polystyrene bead-laden fluid and imaged with transmission mode microscopy. The high quality optical images prove CO2 laser processing as a practical method to reduce the surface roughness of microfluidic channels fabricated by femtosecond laser irradiation. We further compared the traditional and laser-based glass micromachining approaches, which includes FLAC followed by the CO2 polishing technique.

Structural and physicochemical characterization of nanoparticles synthesized from an aqueous extract of wheat bran by a cold-set gelation/desolvation approach

The aqueous extracts of wheat bran are rich in carbohydrates and contain water soluble proteins with a high content of Asp and Glu, which could enable the fabrication of nanoparticles through the cold-set gelation/desolvation of the proteins, while carbohydrates would act as the matrix on which the nanoparticles are formed. In order to test this hypothesis, in this work it was assayed the desolvation of the wheat bran aqueous extracts using CaCl2 as desolvation agent. Aggregates between 3 and 5 μm were formed immediately after addition of a range of CaCl2 concentrations, as detected by dynamic light scattering. Scanning electron microscopy showed that such aggregates consisted of spherical nanoparticles with diameters between 20 and 100 nm, immersed into a matrix composed mainly by polysaccharides. This matrix was disrupted by ultra-sonication and individual nanoparticles, arranged in a necklace fashion, were visible by scanning electron microscopy in transmission mode. This is the first report on the formation of nanoparticles from an aqueous extract of wheat bran by a cold-set gelation/desolvation approach.

Characterization of a Multimodal and Multispectral Led Imager: Application to Organic Polymer’s Microspheres with Diameter Φ = 10.2 μm

Multispectral microscopy enables information enhancement in the study of specimens because of the large spectral band used in this technique. A low cost multimode multispectral microscope using a camera and a set of quasi-monochromatic Light Emitting Diodes (LEDs) ranging from ultraviolet to near-infrared wavelengths as illumination sources was constructed. But the use of a large spectral band provided by non-monochromatic sources induces variation of focal plan of the imager due to chromatic aberration which rises up the diffraction effects and blurs the images causing shadow around them. It results in discrepancies between standard spectra and extracted spectra with microscope. So we need to calibrate that instrument to be a standard one. We proceed with two types of images comparison to choose the reference wavelength for image acquisition where diffraction effect is more reduced. At each wavelength chosen as a reference, one image is well contrasted. First, we compare the thirteen well contrasted images to identify that presenting more reduced shadow. In second time, we determine the mean of the shadow size over the images from each set. The correction of the discrepancies required measurements on filters using a standard spectrometer and the microscope in transmission mode and reflection mode. To evaluate the capacity of our device to transmit information in frequency domain, its modulation transfer function is evaluated. Multivariate analysis is used to test its capacity to recognize properties of well-known sample. The wavelength 700 nm was chosen to be the reference for the image acquisition, because at this wavelength the images are well contrasted. The measurement made on the filters suggested correction coefficients in transmission mode and reflection mode. The experimental instrument recognized the microsphere’s properties and led to the extraction of the standard transmittance and reflectance spectra. Therefore, this microscope is used as a conventional instrument.