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Background: For effective investigation of the developing structure and chemistry of bone, comprehensive studies including compositional analysis can be achieved through the gradual observation from the micro- to nanometer scale via correlative light and electron microscopy (CLEM). This technique is particularly useful considering the complex hierarchical arrangement of bioapatite and collagen fibrils which may vary according to specific bone tissue types (i.e., lamellar bone and woven bone) and different growth stages. Scanning electron microscopy (SEM) accompanied with the attachment of the scanning transmission electron microscopy (STEM) detector, referred to as the STEM-in-SEM can be utilized to produce high contrast images from materials composed of light elements, and efficiently allows the selection of suitable accelerating voltage for energy-dispersive spectroscopy (EDS). This study aims to emphasize the efficacy of CLEM techniques through applying STEM-in-SEM and EDS analyses, and its application to comparative murine bone investigation in differing ontogenetic stages. Findings: We have designed a new grid-holder which can be used for both light and electron microscopy, and we presented an imaging technique for TEM specimens via reflective light microscopy (RLM). For performing CLEM, ultra-thin-sections (UTS) prepared from the femoral bones of 1- to 16-week old of Sprague-Dawley (SD) rats provided light and electron micrographs that can be correlated based on the regions of interest (ROIs). STEM-in-SEM micrographs revealed information not attainable by secondary electron (SE) and back-scattered electron (BSE) micrographs. In addition, for analyzing chemical variation according to growth and development of femoral bones from 1- to 16-week-old rats, comparative chemical analysis was performed through STEM-in-SEM EDS with two reference materials. Conclusion: Herein, from femoral bones of SD rats, we have confirmed the rapid chemical and structural variations within the first 8 weeks after birth. STEM-in-SEM micrographs revealed the bone development process of the early stage porous bone matrix subsequently being filled with collagen fibrils and bioapatite. In addition, chemical analysis for carbon and oxygen showed the ratios of inorganic to organic phases according to growth and progress in bone mineralization. As a result, we were able to postulate the growth mechanism of murine femoral bone in the neonatal stages of development. We also anticipate that our CLEM techniques can be further utilized for more thorough investigation of bone structure and chemistry in diverse scales. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the changes in individual condyles from 5 to 8 years in patients with temporomandibular joint (TMJ) osteoarthritis using 3-dimensional cone beam computed tomography (3D CBCT) reconstruction and superimposition. To assess the longitudinal TMJ changes, CBCT was performed at initial (T0) and final (T2) timepoints that were at least 5 years apart and at a middle (T1) timepoint. To improve the accuracy, we used a novel superimposition method that designated areas of coronoid process and mandibular body. The differences in the resorption and apposition amounts were calculated between each model via maximum surface distances. The greatest resorption and apposition observed were − 7.48 and 2.66 mm, respectively. Evaluation of the changes in each condyle showed that osteoarthritis leads to both resorption and apposition. Resorption was mainly observed in the superior region, while high apposition rates were observed (in decreasing order) in the posterior, lateral, and anterior regions. The medial parts showed greater apposition than the lateral parts in all regions. Our superimposition method reveals that both resorption and apposition were observed in condyles with TMJ osteoarthritis, and resorption/apposition patterns depend on the individual condyle and its sites. [ABSTRACT FROM AUTHOR]

Polyamidoamine (PAMAM) dendrimer is one of the cationic polymers widely used in nonviral vector-based gene therapy. As PAMAM comprises biodegradable peptide bonds, it shows biocompatibility and relatively low cytotoxicity. In comparison with polyethylenimine (PEI, 25 kDa) that shows high transfection efficiency, the native PAMAM dendrimer has limitations as a gene carrier. To improve the gene expression efficiency, we introduced KRTR peptides derived from the influenza B virus nucleoprotein, known as a nuclear localization signal (NLS), to PAMAM generation 3. We synthesized PAMAM-RTRK and PAMAM-HRTRK and evaluated the effects of the histidine residue on cytotoxicity. The transfection efficiency of PAMAM-RTRK and PAMAM-HRTRK was similar to that of PEI in HepG2 cells, while their gene expression capacities were much higher than capacity of PEI in A549 cells. PAMAM-HRTRK showed relatively lower cytotoxicity than PAMAM-RTRK in the two cell lines, but the transfection capacity was similar for the two polymers. These results imply that the combination of histidine and KRTR peptide may potentially result in a novel PAMAM-based vector that could be used for prolonged gene therapy through the reduction in cytotoxicity and enhancement of gene expression. [ABSTRACT FROM AUTHOR]

The GaN thin film deposited on an amorphous glass substrate was analyzed by using transmission electron microscopy with a new automated crystal orientation mapping tool. Film deposition was made at 600°C for 4 h by the hyperthermal neutral beam (HNB) source. Columnar crystals oriented to the [0001] direction without significant disordering were clearly observed. Electron diffraction patterns indicated that the crystals have mainly two different zone axes, [2 $\bar 1$ $\bar 1$0] and [10 $\bar 1$0]. This crystallographic and microstructural information provides the guidance for future works for the HNB source to obtain GaN thin films of higher quality on amorphous substrates. [ABSTRACT FROM AUTHOR]

A sub-Å resolution analysis of a Si crystal structure was performed successfully using a phase extension technique that combined the high-voltage electron microscopy (HVEM) and energy-filtered precession electron diffraction (EF-PED) methods. The high resolution electron microscopy (HREM) image of Si [211] was obtained with a spatial resolution of 0.12 nm using HVEM. The electron diffraction of Si [211] was acquired with a spatial resolution of 0.06 nm by employing the EF-PED method. The phase information of Si [211] was extracted from a deconvoluted image by applying instrumental parameters. Its amplitude information with higher spatial resolution was quasi-kinematically extracted from EF-PED data. Finally, it was revealed that Si atoms were clearly separated with a resolution of 0.78 Å in the enhanced resolution image. Therefore, it is expected that the combination of the HVEM and EF-PED methods will be a useful and powerful tool for the quantitative characterization of nanostructured materials. [ABSTRACT FROM AUTHOR]

A dorsal rib portion from the post-cranial skeleton of a small ornithischian dinosaur discovered from the fossiliferous locality in Boseong was analyzed through Optical Microscopy (OM), Scanning Electron Microscopy (SEM), Electron Probe Microanalyser (EPMA), X-ray Diffraction (XRD), and Transmission Electron Microscopy (TEM) to determine the detailed microstructure and components of the fossilized dinosaur bone. The rib bone portion was specifically chosen as an initial research sample to establish efficient experimental methodology in order to apply to future dinosaur osteohistological study. Since the fossilized bone was highly compressed by the surrounding matrix, distinct features of bone tissues were not clearly visible in OM cross sections. Instead, we observed two other features: (1) numerous patches of calcite crystals in various orientations filling the void region; (2) apatite crystals of 10∼200 nm size constituting the bone matrix region, which is revealed by XRD, EPMA, SEM, and TEM. The data we have obtained so far is preliminary to directly elucidate the specific microstructural properties of fossilized bone such as bone formation and growth patterns, but we have provided possibility of revealing the characteristic features of dinosaur bone microstructure in nano-scale and established efficient specimen preparation methods for correlative Optical Microscopy (OM)-Electron Microscopy (EM) study. [ABSTRACT FROM AUTHOR]

Background: With the application of the scanning transmission electron microscopy (STEM) detector, transmitted electron images similar to transmission electron microscopy (TEM) can be obtained from scanning electron microscopy (SEM), which is referred to STEM-in-SEM imaging. Compared to TEM-energy dispersive spectroscopy (EDS), SEM-EDS is expected to be more efficient and reliable for chemical analysis of TEM specimens due to the larger sample space in SEM and the higher take-off angle of the SEM-EDS detector. Unfortunately, this advantage is not well applied to TEM specimens in practice, mainly because of fault signals generated from the commercial grid holders used in SEM and STEM-in-SEM. This study is designed to solve this problem by modifying the commercial holders and to test them through EDS analysis of apatite phases. Findings: We first changed the way of assembling parts of the commercial multi-grid holder for SEM. This new assembly was capable of producing a thinner upper cover part, resulting in the reduction of the EDS fault signals generated from the holder. Furthermore, the thin upper part allowed us to get images in shorter working distances, that is, at higher magnifications. This design concept was also applied to the commercial single-grid holder for STEM-in-SEM, producing a new multi-grid holder to be used for loading multiple samples. Conclusion: We confirmed that the new holders produced reliable chemical data from apatite phases. In case of oxygen analysis, despite of low electron brightness from the tungsten source, 5 kV provided more stable acquisition and signal yields than 15 kV. We expect that these modified holders facilitate more efficient EDS analysis for multiple samples under the same analytical conditions in SEM and STEM-in-SEM. [ABSTRACT FROM AUTHOR]

Osteohistological researches on dinosaurs are well documented, but descriptions of direct correlations between the bone microstructure and corresponding nanostructure are currently lacking. By applying correlative microscopy, we aimed to verify that well-preserved osteohistological features correlate with pristine fossil bone nanostructures from the femoral bones of Koreanosaurus boseongensis. The quality of nanostructural preservation was evaluated based on the preferred orientation level of apatite crystals obtained from selected area electron diffraction (SAED) patterns and by measuring the 'arcs' from the {100} and {002} diffraction rings. Unlike our expectations, our results revealed that well-preserved microstructures do not guarantee pristine nanostructures and vice versa. Structural preservation of bone from macro- to nanoscale primarily depends on original bioapatite density, and subsequent taphonomical factors such as effects from burial, pressure, influx of external elements and the rate of diagenetic alteration of apatite crystals. Our findings suggest that the efficient application of SAED analysis opens the opportunity for comprehensive nanostructural investigations of bone. [ABSTRACT FROM AUTHOR]