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Delivery of liposomal doxorubicin (Doxil) in a breast cancer tumor model: investigation of potential enhancement by pulsed-high intensity focused ultrasound exposure.

RATIONALE AND OBJECTIVES: To investigate the potential of using pulsed high-intensity focused ultrasound (HIFU) exposures to enhance the delivery, and hence therapeutic effect of liposomal doxorubicin (Doxil) in a murine breast cancer tumor model. MATERIALS AND METHODS: Tumors were grown in the bilateral flanks of mice using a mammary adenocarcinoma cell line. Experiments consisted of exposing one of two tumors to pulsed-HIFU, followed by tail vein injections of Doxil. Tumor growth rates were monitored, and assays carried out for doxorubicin concentration in these tumors as well as in a second (squamous cell carcinoma) tumor model and in muscle. Laser scanning confocal microscopy was used with fluorescent probes to observe both the uptake of polystyrene nanoparticles and dilation of exposed blood vessels. Additional experiments involving histologic analysis and real-time temperature measurements were performed to determine the safety of the exposures. RESULTS: Pulsed-HIFU exposures were shown to be safe, producing no apparent deleterious effects in the tumors. The exposures, however, were not found to enhance the delivery of Doxil, and consequently did not allow for lower doses for obtaining tumor regression. Imaging with a fluorescent dextran showed blood vessels to be dilated as a result of the exposures. Experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumors. CONCLUSION: Although past studies have shown the advantages of pulsed-HIFU exposures for enhancing delivery, this was not observed with the liposomes, apparently because of their inherent ability to preferentially accumulate into tumors on their own. Potential mechanisms for enhanced uptake of non-liposomal nanoparticles are discussed.

Adenocarcinoma↗

Possible mechanism of polycation liposome (PCL)-mediated gene transfer.

A novel gene transfer system utilizing polycation liposomes (PCLs), obtained by modifying liposomes with cetyl polyethylenimine (PEI), was previously developed (Gene Ther. 7 (2002) 1148). PCLs show notable transfection efficiency with low cytotoxicity. However, the mechanism of PCL-mediated gene transfer is still unclear. In this study, we examined the intracellular trafficking of PCL-DNA complexes by using HT1080 cells, fluorescent probe-labeled materials, and confocal laser scan microscopy. We found that the PCL-DNA complexes were taken up into cells by the endosomal pathway, since both cellular uptake of the complex and gene expression were blocked by wortmannin, an inhibitor of this pathway. We also observed that the plasmid DNA and cetyl PEI complex became detached from the PCL lipids and was preferentially transferred into the nucleus in the form of the complex, whereas the PCL lipids remained in the cytoplasmic area, possibly in the endosomes. In fact, nigericin, which dissipates the pH gradient across the endosomal membrane, inhibited the detachment of lipids from the PCL-DNA complex and subsequent gene expression. Taken together, our data indicate the following mechanism for gene transfer by PCLs: PCLs effectively transfer DNA to endosomes and release cetyl PEI-DNA complexes into the cytosol. Furthermore, cetyl PEI also contributes to gene entry into the nucleus.

Androstadienes↗

Layer-by-layer films of hemoglobin or myoglobin assembled with zeolite particles: electrochemistry and electrocatalysis.

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming [zeolite/protein](n) layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about -0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)) in solution were catalytically reduced at [zeolite/protein](7) film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in [zeolite/protein](7) films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)(6)(3-) as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of ]zeolite/protein](n) films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.

Adsorption↗

Experimental studies on the effect of osteotomy technique on the bone regeneration in distraction osteogenesis.

The objective of the experiment was to study the effect of osteotomy technique on the course of bone regeneration in distraction osteogenesis in sheep. Thirty five merino sheep were divided into 5 groups of 7 sheep each, depending on the osteotomy technique: subcutaneous corticotomy, open corticotomy, Cattaneo corticotomy, open osteotomy and osteoclasia. Formation of regenerate and its remodeling was assessed, based on roentgenograms and morphological examination (bone sections, histological evaluation, SEM, superficial X-ray analysis). Irrespective from the osteotomy technique applied, in all experimental groups, regenerates filling the distraction gap were obtained. Analysis of bone sections and histological slides indicated the most advanced osteogenesis processes in the osteoclasia group. SEM examination and X-ray microanalysis confirmed not only the presence of mature bone tissue structures in this group, but also showed a mineralization close to that of a healthy bone. Osteogenesis was the slowest in the group of subcutaneous corticotomy. The observed histological picture and analysis of elemental composition indicate a normal course of distraction osteogenesis in this group, but its consecutive phases proceed with a certain delay, compared to the remaining groups. With a good deal of caution, it can be assumed that the delay of osteogenesis in the group of subcutaneous corticotomy in the 14th post-operative week was ca. 3 weeks compared to the osteoclasia group. In all experimental groups, few cartilaginous nodules were observed in regenerates 5 weeks and, in few cases, 9 weeks old. Ossification in such places was indirect, cartilage-based. In the remaining regions direct, membrane-based ossification prevailed. Direct apposition bone lamellae on islands of fibrous bone tissue was observed in regenerates aged 9 and 14 weeks, especially in periosteal regions. The intensity and rate of formation of new bone tissue varied between the experimental groups, based on roentgenological analysis and histological evaluation. Complex morphological studies on the course of regeneration in sheep showed the most advanced remodeling and mineralization of bone tissue in animals subject to osteoclasia.

Animals↗

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells.

As amply documented by electrophysiology, depolarisation in Paramecium induces a Ca(2+) influx selectively via ciliary voltage-dependent Ca(2+)-channels, thus inducing ciliary beat reversal. Subsequent downregulation of ciliary Ca(2+) has remained enigmatic. We now analysed this aspect, eventually under overstimulation conditions, by quenched-flow/cryofixation, combined with electron microscope X-ray microanalysis which registers total calcium concentrations, [Ca]. This allows to follow Ca-signals within a time period (> or =30ms) smaller than one ciliary beat ( approximately 50ms) and beyond. Particularly under overstimulation conditions ( approximately 10(-5)M Ca(2+) before, 0.5mM Ca(2+) during stimulation) we find in cilia a [Ca] peak at approximately 80ms and its decay to near-basal levels within 110ms (90%) to 170ms (100% decay). This [Ca] wave is followed, with little delay, by a [Ca] wave into subplasmalemmal Ca-stores (alveolar sacs), culminating at approximately 100ms, with a decay to original levels within 170ms. Also with little delay [Ca] slightly increases in the cytoplasm below. This implies rapid dissipation of Ca(2+) through the ciliary basis, paralleled by a rapid, transient uptake by, and release from cortical stores, suggesting fast exchange mechanisms to be analysed as yet. This novel type of coupling may be relevant for some phenomena described for other cells.

Animals↗

Hydrodynamic flow in capillary-channel fiber columns for liquid chromatography.

The flow characteristics of capillary-channel polymer (C-CP) fiber liquid chromatographic (LC) columns have been investigated. The C-CP fibers are manufactured with eight longitudinal grooves (capillary channels) extending the length of the fibers. Three C-CP fiber examples were studied, with fiber dimensions ranging from approximately 35 microm to 65 microm, and capillary-channel dimensions ranging from approximately 6 microm to 35 microm. The influence of fiber packing density and column inner diameter on peak asymmetry, peak width, and run-to-run reproducibility have been studied for stainless steel LC columns packed with polyester (PET) and polypropylene (PP) C-CP fibers. The van Deemter A-term was evaluated as a function of fiber packing density (approximately 0.3 g/cm(3)-0.75 g/cm(3)) for columns of 4.6 mm inner diameter (i.d.) and at constant packing densities for 1.5 mm, 3.2 mm, 4.6 mm, and 7.7 mm i.d. columns. Although column diameter had little influence on the eluting peak widths, peak asymmetry increased with increasing column diameter. The A-terms for the C-CP fiber packed columns are somewhat larger than current commercial, microparticulate-packed columns, and means for improvement are discussed. Applications in the area of protein (macromolecule) separations appear the most promising at this stage of the system development.

Chromatography, Liquid↗

SEM-EDS probing of morphological and physiological changes produced by a porphyrin photosensitizer in Psammobatis extenta electrocytes.

Morphological and physiological changes produced by the intracellular localization of an alkyl long-chain tetraphenylporphyrin photosensitizer in Psammobatis extenta electrocytes were studied by means of SEM-EDS. Immediately after photosensitizer penetration, electrocytes swell and their convex faces lose all invaginations. This effect is due to chloride and sodium ion flux into electrocytes, in accordance with energy dispersive X-ray microanalysis.

Animals↗

Raman microscopy of selected tungstate minerals.

A series of tungstate bearing minerals including scheelite, stolzite, ferberite, hübnerite, wolframite, russellite, tungstenian wulfenite and cuprotungstite have been analyzed by Raman microscopy. The results of the Raman spectroscopic analysis are compared with published data. These minerals are closely related and often have related paragenesis. Raman microscopy enables the selection of individual crystals of these minerals for spectroscopic analysis even though several of the minerals can be found in the same matrix because of the pargenetic relationships between the minerals. The Raman spectra are assigned according to factor group analysis and related to the structure of the minerals. These minerals have characteristically different Raman spectra. The nu1(Ag) band is observed at 909 cm(-1) and although the corresponding nu1(Bu) vibration should be inactive a minor band is observed around 894 cm(-1). The bands at 790 and 881 cm(-1) are associated with the antisymmetric and symmetric Ag modes of terminal WO2. The band at 695 cm(-1) is interpreted as an antisymmetric bridging mode associated with the tungstate chain. The nu4(Eg) band was absent for scheelite. The bands at 353 and 401 cm(-1) are assigned as either deformation modes or as r(Bg) and delta(Ag) modes of terminal WO2. The band at 462 cm(-1) has an equivalent band in the infrared at 455 cm(-1) assigned as delta(as)(Au) of the (W2O4)n chain. The band at 508 cm(-1) is assigned as nu(sym)(Bg) of the (W2O4)n chain.

Electron Probe Microanalysis↗

Raman spectroscopic study of the molecular structure of the uranyl mineral zippeite from Jáchymov (Joachimsthal), Czech Republic.

Raman spectra at 298 and 77K and infrared spectra of the uranyl sulfate mineral zippeite from Jáchymov (Joachimsthal), Czech Republic, K(0.6)(H(3)O)0.4[(UO(2))6(SO(4))3(OH)7].8H2O, were studied. Observed bands were tentatively attributed to the (UO(2))2+ and (SO(4))2- stretching and bending vibrations, the OH stretching vibrations of water molecules, hydroxyls and oxonium ions, and H(2)O, oxonium, and delta U-OH bending vibrations. Empirical relations were used for the calculation of U-O bond lengths in uranyl R (A)=f(nu(3) or nu(1)(UO(2))2+). Calculated U-O bond lengths are in agreement with U-O bond lengths from the single crystal structure analysis and those inferred for uranyl anion sheet topology of uranyl pentagonal dipyramidal coordination polyhedra. The number of observed bands supports the conclusion from single crystal structure analysis that at least two symmetrically distinct U6+ (in uranyls) and S6+ (in sulfates), water molecules and hydroxyls may be present in the crystal structure of the zippeite studied. Strong to very weak hydrogen bonds present in the crystal structure of zippeite studied were inferred from the IR spectra.

Czech Republic↗

Distributions of calcium in A and I bands of skinned vertebrate muscle fibers stretched to beyond filament overlap.

Measurements were made of the distributions of total calcium along the length of A and I bands in skinned frog semitendinosus muscles using electron probe x-ray microanalysis. Since calcium in the water space was kept below the detection limit of the technique, the signal was assumed to reflect the distribution of calcium bound to myofilament proteins. Data from sarcomeres with overlap between thick and thin filaments showed enhancement of calcium in this region, as previously demonstrated in rabbit psoas muscle fibers in rigor (Cantino, M. E., T. S. Allen, and A. M. Gordon. 1993. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys. J. 64:211-222). Such enhancement could arise from intrinsic non-uniformities in calcium binding to either thick or thin filaments or from enhancement of calcium binding to either filament by rigor cross-bridge attachment. To test for intrinsic variations in calcium binding, calcium distributions were determined in fibers stretched to beyond filament overlap. Calcium binding was found to be relatively uniform along both thick and thin filaments, and therefore cannot account for the increased calcium observed in the overlap region. From these results it can be concluded that the observed enhancement of calcium is due to an increase in calcium binding to myofilaments as a result of rigor attachment of cross-bridges to actin. The source of the enhancement is most likely an increase in calcium binding to troponin, although enhancement of calcium binding to myosin light chains cannot be ruled out.

Animals↗

Characterisation of cement pastes by inverse gas chromatography.

Two cement pastes, commonly used in concrete formulations, were characterised by IGC at 35-80 degrees C before and after coating with an epoxy resin and a hardener. The cements are mixtures of hydrates in various proportions, such as calcium silicate hydrate (CaO-SiO2-H2O) and calcium hydroxide Ca(OH)2. Apolar and polar probes were used to determine the dispersive and acid-base characteristics of the cement pastes. These materials have high surface energy as judged from the dispersive contribution to the surface free energy (gamma(s)d) values lying in the 50-70 mJ/m2 range at 60-80 degrees C. Examination of the specific interactions permitted to show that the cement pastes are strongly amphoteric species with a substantial predominant Lewis basicity that is in line with the basic pH of their aqueous suspensions. Following coating with an epoxy resin (DGEBA) and a hardener (triethylene tetramine), the surface energy of the cements decreases substantially with the mass loading of the organic material. The surface thermodynamic properties were also correlated with the surface chemical composition as determined by X-ray photoelectron spectroscopy.

Chromatography, Gas↗

Distribution and activity of ammonia oxidizing bacteria in a large full-scale trickling filter.

The biofilm in a full-scale nitrifying trickling filter (NTF) treating municipal wastewater has been investigated with microbiological methods using fluorescence in situ hybridization (FISH) with 16S rRNA oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM) and mathematical modeling using a dynamic multi-species biofilm reactor model. Ammonia oxidizing bacteria (AOB) were found to belong to the genus Nitrosomonas at different depths in the NTF at every sampling occasion, corresponding to different long-term operational conditions for the NTF. Both the measurements and the corresponding simulated predictions showed the same general trend of a decrease with filter depth of the amount of biofilm, the proportion of AOB to all bacteria and the total amount of AOB. The latter decreased by several times from top to bottom of the NTF. Measurements and simulations of potential ammonium oxidizing activity in the biofilm also showed a decreasing activity with depth in the NTF, which generally was operating at close to complete nitrification. However, no difference was observed when the activity was normalized to the amount of biofilm, despite decreasing proportions of AOB to all bacteria with depth in the NTF. This could be explained by diffusion limitations in the biofilm from the upper parts of the NTF according to the biofilm reactor model. The relatively good agreement between the simulations and the measurements shows that the kind of biofilm reactor model used can qualitatively describe an averaged behavior and averaged composition of the biofilm in the reactor.

Ammonia↗

Porcelain adherence to dental cast CP titanium: effects of surface modifications.

OBJECTIVES: A reaction layer forms on cast titanium surfaces due to the reaction of the molten titanium with the investment material. Such a layer prevents strong adhesion between titanium and porcelain. This study characterized the effects of surface modifications on cast titanium surfaces and titanium-ceramic adhesion. METHODS: ASTM grade II CP titanium was cast into an MgO-based mold. Castings were devested by sandblasting with alumina particles, and subjected to surface modification by immersion in one of the following solutions: (1) 35% HNO3-5% HF at room temperature for 1min; (2) 50% NaOH-10% CuSO4 x 5H2O at 105 degreesC for 10min; (3) the NaOH-CuSO4 solution followed by the HNO3-HF solution; (4) 50% NaOH-10% NaSO4 at 105 degreesC for 10 min; (5) the NaOH-NaSO4 solution followed by the HNO3-HF solution; and (6) 50% NaOH solution at 105 degreesC for 10min. Surfaces only sandblasted with alumina were used as controls. Specimen surfaces were characterized by XRD and SEM/EDS, and hardness-depth profiles were determined. All specimens were sandblasted with 110 microm alumina particles before porcelain firing. An ultra-low-fusing porcelain (Vita Titankeramik) was fused on the titanium surfaces. The titanium-ceramic adhesion was characterized by a biaxial flexure test, and area fraction of adherent porcelain (AFAP) was determined by X-ray spectroscopy. RESULTS: EDS analyses revealed a substantial amount (13-17 wt%) of Al on the control, and specimens modified with Methods 2, 4, and 6. XRD revealed residual stress in the titanium surfaces and corundum on the control, and Methods 2, 4, and 6 specimens. A new Ti(Cu, Al)2 phase was identified on the titanium surfaces modified by immersion in 50% NaOH-10% CuSO4 x 5H2O aqueous solution. Reduced residual stress was observed on Method 1, 3, and 5 specimens. No corundum peaks were detected on these specimens. Compared to the control, significantly lower (P < 0.05) hardness values were found for Methods 1-3 and Method 5 specimens at 20 microm below the surfaces and for Method 1-5 specimens at 50 microm below the surfaces. Significantly higher (P < 0.05) AFAP values were found for surfaces modified with Methods 2-6 compared to the control and Method 1, and no significant differences were found among Methods 1-6, and between control and Method 1. SIGNIFICANCE: Based on the results from the present study, porcelain adherence to cast CP titanium can be improved by the caustic baths used in the study.

Dental Casting Investment↗

Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury.

Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral-central nervous system pathobiology, and, particularly, alcohol-induced strokes.

Alcoholism↗

Application of quantitative analytical electron microscopy to the mineral content of insect cuticle.

Quantification of calcium in the cuticle of the fly larva Exeretonevra angustifrons was undertaken at the micron scale using wavelength dispersive X-ray microanalysis, analytical standards, and a full matrix correction. Calcium and phosphorus were found to be present in the exoskeleton in a ratio that indicates amorphous calcium phosphate. This was confirmed through electron diffraction of the calcium-containing tissue. Due to the pragmatic difficulties of measuring light elements, it is not uncommon in the field of entomology to neglect the use of matrix corrections when performing microanalysis of bulk insect specimens. To determine, firstly, whether such a strategy affects the outcome and secondly, which matrix correction is preferable, phi-rho (z) and ZAF matrix corrections were contrasted with each other and without matrix correction. The best estimate of the mineral phase was found to be given by using the phi-rho (z) correction. When no correction was made, the ratio of Ca to P fell outside the range for amorphous calcium phosphate, possibly leading to flawed interpretation of the mineral form when used on its own.

Animals↗

The determination of the efficiency of energy dispersive X-ray spectrometers by a new reference material.

A calibration procedure for the detection efficiency of energy dispersive X-ray spectrometers (EDS) used in combination with scanning electron microscopy (SEM) for standardless electron probe microanalysis (EPMA) is presented. The procedure is based on the comparison of X-ray spectra from a reference material (RM) measured with the EDS to be calibrated and a reference EDS. The RM is certified by the line intensities in the X-ray spectrum recorded with a reference EDS and by its composition. The calibration of the reference EDS is performed using synchrotron radiation at the radiometry laboratory of the Physikalisch-Technische Bundesanstalt. Measurement of RM spectra and comparison of the specified line intensities enables a rapid efficiency calibration on most SEMs. The article reports on studies to prepare such a RM and on EDS calibration and proposes a methodology that could be implemented in current spectrometer software to enable the calibration with a minimum of operator assistance.

Journal Article↗

Raman imaging and Kelvin probe microscopy for the examination of the heterogeneity of doping in polycrystalline boron-doped diamond electrodes.

The issue of the heterogeneity of boron doping in microcrystalline diamond films was addressed by four different methods: micro-Raman spectroscopy and Raman imaging, Kelvin probe force microscopy, conducting atomic force microscopy, and scanning electrochemical microscopy. The samples were commercially available films from Windsor Scientific, with an average boron concentration of about 5 x 10(20) cm(-3). In agreement with previous works, all of the methods showed that the boron uptake was nonuniform across the surface of the electrode. Two different types of regions were evidenced, with metallic or semiconducting properties that were characterized with different types of Raman spectra. The line shape of these spectra was strongly dependent on the excitation wavelength. Local variations in electroactivity were evidenced by the SECM curves, which are related to the electronic properties of the individual grains, which, in turn, are governed by the boron content of the individual crystallites. In this study, two different micro-Raman imaging techniques were used that reveal the grain structure of the films: the images constructed from the diamond line intensity perfectly reproduced the optical image obtained by illuminating the sample in reflection. The method also allows detection of the presence of nondiamond carbon, especially in the metallic parts of the samples. Other spectral features (intensity of the boron-related broad lines, as well as the frequency and width of the diamond line) were used to construct images. In every case, the grain structure of the film was revealed, as well as twinning within individual crystallites. All approaches revealed that no enhanced doping or boron depletion occurred at the grain boundaries.

Journal Article↗