Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.     

Automated segmentation of wood fibres in micro‐CT images of paper

A novel algorithm has been developed and validated to isolate individual papermaking fibres in micro‐computed tomographic images of paper handsheets as a first step to characterize the structure of the paper. The three‐step fibre segmentation algorithm segments the papermaking fibres by (i) tracking the hollow inside the fibres via a modified connected component methodology, (ii) extracting the fibre walls using a distance transform and (iii) labelling the fibres through collapsed sections by a final refinement step. Furthermore, postprocessing algorithms have been developed to calculate the length and coarseness of the segmented fibres. The fibre segmentation algorithm is the first ever reported method for the automated segmentation of the tortuous three‐dimensional morphology of papermaking fibres within microstructural images of paper handsheets. The method is not limited to papermaking fibres, but can be applied to any material consisting of tortuous and hollow fibres.     

Application of micro‐CT: A new method for stone drilling research

Drilling is one of the most complex techniques for making ancient stone implement or adornment. For smaller perforations, it is very difficult to effectively observe drilling marks under microscope, SEM, or to obtain negative silicone rubber cast. In this report, a new exclusive nondestructive method was first introduced to resolve the observation difficulty. Virtual 3D reconstruction by using micro CT (μCT) was successfully applied to disclose drilling tool marks on the inner wall of one small perforation and its relative drilling technique was deduced, which implied that μCT has great potential to understand ancient stone drilling. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Stress and strain distribution in demineralized enamel: A micro‐CT based finite element study

Physiological oral mechanical forces may play a role on the progression of enamel carious lesions to cavitation. Thus, the aim of this study was to describe, by 3D finite element analysis, stress, and strain patterns in sound and carious enamel after a simulated occlusal load. Micro‐CT based models were created and meshed with tetrahedral elements (based on an extracted third molar), namely: a sound (ST) and a carious tooth (CT). For the CT, enamel material properties were assigned according to the micro‐CT gray values. Below the threshold corresponding to the enamel lesion (2.5 g/cm³) lower and isotropic elastic modulus was assigned (E = 18 GPa against E₁ = 80 GPa, E₂ = E₃ = 20 GPa for sound enamel). Both models were imported into a FE solver where boundary conditions were assigned and a pressure load (500 MPa) was applied at the occlusal surface. A linear static analysis was performed, considering anisotropy in sound enamel. ST showed a more efficient transfer of maximum principal stress from enamel to the dentin layer, while for the CT, enamel layer was subjected to higher and concentrated loads. Maximum principal strain distributions were seen at the carious enamel surface, especially at the central fossa, correlating to the enamel cavity seen at the original micro‐CT model. It is possible to conclude that demineralized enamel compromises appropriate stress transfer from enamel to dentin, contributing to the odds of fracture and cavitation. Enamel fracture over a dentin lesion may happen as one of the normal pathways to caries progression and may act as a confounding factor during clinical diagnostic decisions. Microsc. Res. Tech. 78:865–872, 2015. © 2015 Wiley Periodicals, Inc.     

A fast experimental beam hardening correction method for accurate bone mineral measurements in 3D μCT imaging system

Bone mineral density plays an important role in the determination of bone strength and fracture risks. Consequently, it is very important to obtain accurate bone mineral density measurements. The microcomputerized tomography system provides 3D information about the architectural properties of bone. Quantitative analysis accuracy is decreased by the presence of artefacts in the reconstructed images, mainly due to beam hardening artefacts (such as cupping artefacts). In this paper, we introduced a new beam hardening correction method based on a postreconstruction technique performed with the use of off‐line water and bone linearization curves experimentally calculated aiming to take into account the nonhomogeneity in the scanned animal. In order to evaluate the mass correction rate, calibration line has been carried out to convert the reconstructed linear attenuation coefficient into bone masses. The presented correction method was then applied on a multimaterial cylindrical phantom and on mouse skeleton images. Mass correction rate up to 18% between uncorrected and corrected images were obtained as well as a remarkable improvement of a calculated mouse femur mass has been noticed. Results were also compared to those obtained when using the simple water linearization technique which does not take into account the nonhomogeneity in the object.     

Establishing the archaeo‐metallurgic ornamentation process of an axe from the bronze age by OM,SEM‐EDX,and Micro‐FTIR

Our article presents the results of the analyses we performed by corroborating the Optical Microscopy, Scanning Electron Microscopy, Energy Dispersive Xray Analysis and micro Fourier Transformed InfraRed Analysis techniques to identify the archaeo‐metallurgic casting and ornamentation procedure of a decorated disk‐butted axe, which was discovered recently east of the Carpathian mountains, in the Moldavian Plateau. There are few known axes of that type found (A1, according to the usual typologies), as they are specific to the Middle Bronze Age period west of the Carpathians—the Wietenberg, Suciu de Sus, and Otomani‐Füzesabony cultures. The experimental data on the item under study revealed the fact that after casting it in molds made from porous silicone‐based stone, the object was coated with a thin layer, by immersing it in a lightly fusible tin alloy, whose main alloy component was copper and arsenic and iron as secondary components. After refining the shiny white layer, they applied a beautiful decoration pattern made by incision and engraving. This battle axe was an indication of higher status, such items usually being owned by community leaders. This important fact proves that the ancient metallurgic craftsmen were able to elaborate and manufacture various alloys from which they made beautiful objects and it also offers a new insight into the social and symbolic function of certain antique bronze items. Microsc. Res. Tech. 77:918–927, 2014. © 2014 Wiley Periodicals, Inc.     

Virtual histology by means of high‐resolution X‐ray CT

Micro‐CT is a non‐destructive technique for 3D tomographic investigation of an object. A 3D representation of the internal structure is calculated based on a series of X‐ray radiographs taken from different angles. The spatial resolution of current laboratory‐used micro‐CT systems has come down over the last years from a few tens of microns to a few microns. This opens the possibility to perform histological investigations in 3D on a virtual representation of a sample, referred to as virtual 3D histology. The advantage of micro‐CT based virtual histology is the immediate and automated 3D visualization of the sample without prior slicing, sample preparation like decalcification, photographing and aligning. This not only permits a drastic reduction in preparation time but also offers the possibility to easily investigate objects that are difficult to slice. This article presents results that were obtained on punch biopsies of horse skin, (dental) alveolus of ponies and chondro‐osseous samples from the tarsus of foals studied with the new high resolution micro‐CT set‐up (HRXCT) at the Ghent University (Belgium) ( http://www.ugct.ugent.be ). This state‐of‐the‐art set‐up provides a 1 micron resolution and is therefore ideally suited for a direct comparison with standard light microscopy–based histology.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

X‐ray CT and histological imaging of xylem vessels organization in Mimosa pudica

Mimosa pudica has three distinct specialized organs, namely, pulvinus, secondary pulvinus, and pulvinule, which are respectively controlling the movements of petioles, leaflets, and pinna in response to external stimuli. Water flow is a key factor for such movements, but detailed studies on the organization of the vascular system for water transport in these organs have not been published yet. In this study, organizations of the xylem vessels and morphological features of the pulvinus, the secondary pulvinus, and the pulvinule were experimentally investigated by X‐ray computed tomography and histological technique. Results showed that the xylem vessels were circularly distributed in the specialized motile organs and reorganized into distinct vascular bundles at the extremities. The number and the total cross‐sectional area of the xylem vessels were increased inside the specialized motile organs. Morphological characteristics obtained in this study provided new insight to understand the functions of the vascular networks in the dynamic movements of M. pudica. Microsc. Res. Tech. 76:1204–1212, 2013. © 2013 Wiley Periodicals, Inc.     

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐dielectric barrier discharge plasma

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro‐dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro‐DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro‐DBD plasma for various exposure times. The elasticity of a single cell was determined by force–distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma‐treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro‐DBD plasma were related to cell mechanical changes.