FIB-SEM dataset of a human bone osteosarcoma epithelial cell (U2-OS)
Ontology highlight
ABSTRACT:
SUBMITTER: Eija Jokitalo
PROVIDER: EMPIAR-11746 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Accelerating data sharing and reuse in volume electron microscopy.
Czymmek Kirk James KJ Belevich Ilya I Bischof Johanna J Mathur Aastha A Collinson Lucy L Jokitalo Eija E
Nature cell biology 20240401 4
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Project description:Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.
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Project description:Osteosarcoma is the most common primary malignancy of the bone. There have been some advances in surgical and chemotherapeutic strategies, but it is still a tumor with a high mortality rate in children and young adults. Mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) pathway plays an essential role in the development and progression of various tumors. ERK1/2 is a key component of this pathway and hyperactivated in different tumors including osteosarcoma. This study aimed to investigate whether downregulation of ERK1/2 by siRNA (small interfering RNA) could inhibit cell proliferation and invasion and increase chemosensitivity to cisplatin in human osteosarcoma U2-OS cells in vitro. Results showed that the downregulation of ERK1/2 expression by siRNA in human osteosarcoma cells significantly inhibited cell proliferation and invasion in vitro. Furthermore, ERK1/2 knockdown led to cell arrest in the G1/G0 phase of the cell cycle, and eventual apoptosis and chemosensitivity enhancement in tumor cells. Our data reveal that RNAi-mediated downregulation of ERK1/2 expression can lead to potent antitumor activity and chemosensitizing effects in human osteosarcoma.
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Project description:MotivationFIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses.ResultsWith the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions.Availability and implementationSEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.
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Project description:We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non-epithelial spaces (stroma) of many organs (see www.telocytes.com). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB-SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB-SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) - the podoms. FIB-SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.
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Project description:During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam-scanning electron microscopy (cryo-FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium-rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back-scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s-1 is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium-binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns.
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Project description:Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.
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