A joint alignment and reconstruction algorithm for electron tomography to visualize in-depth cell-to-cell interactions.

Electron tomography allows one to obtain 3D reconstructions visualizing a tissue's ultrastructure from a series of 2D projection images. An inherent problem with this imaging technique is that its projection images contain unwanted shifts, which must be corrected for to achieve reliable reconstructions. Commonly, the projection images are aligned with each other by means of fiducial markers prior to the reconstruction procedure. In this work, we propose a joint alignment and reconstruction algorithm that iteratively solves for both the unknown reconstruction and the unintentional shift and does not require any fiducial markers. We evaluate the approach first on synthetic phantom data where the focus is not only on the reconstruction quality but more importantly on the shift correction. Subsequently, we apply the algorithm to healthy C57BL/6J mice and then compare it with non-obese diabetic (NOD) mice, with the aim of visualizing the attack of immune cells on pancreatic beta cells within type 1 diabetic mice at a more profound level through 3D analysis. We empirically demonstrate that the proposed algorithm is able to compute the shift with a remaining error at only the sub-pixel level and yields high-quality reconstructions for the limited-angle inverse problem. By decreasing labour and material costs, the algorithm facilitates further research directed towards investigating the immune system's attacks in pancreata of NOD mice for numerous samples at different stages of type 1 diabetes.

An ultrastructural 3D reconstruction method for observing the arrangement of collagen fibrils and proteoglycans in the human aortic wall under mechanical load.

An insight into changes of soft biological tissue ultrastructures under loading conditions is essential to understand their response to mechanical stimuli. Therefore, this study offers an approach to investigate the arrangement of collagen fibrils and proteoglycans (PGs), which are located within the mechanically loaded aortic wall. The human aortic samples were either fixed directly with glutaraldehyde in the load-free state or subjected to a planar biaxial extension test prior to fixation. The aortic ultrastructure was recorded using electron tomography. Collagen fibrils and PGs were segmented using convolutional neural networks, particularly the ESPNet model. The 3D ultrastructural reconstructions revealed a complex organization of collagen fibrils and PGs. In particular, we observed that not all PGs are attached to the collagen fibrils, but some fill the spaces between the fibrils with a clear distance to the collagen. The complex organization cannot be fully captured or can be severely misinterpreted in 2D. The approach developed opens up practical possibilities, including the quantification of the spatial relationship between collagen fibrils and PGs as a function of the mechanical load. Such quantification can also be used to compare tissues under different conditions, e.g., healthy and diseased, to improve or develop new material models. STATEMENT OF SIGNIFICANCE: The developed approach enables the 3D reconstruction of collagen fibrils and proteoglycans as they are embedded in the loaded human aortic wall. This methodological pipeline comprises the knowledge of arterial mechanics, imaging with transmission electron microscopy and electron tomography, segmentation of 3D image data sets with convolutional neural networks and finally offers a unique insight into the ultrastructural changes in the aortic tissue caused by mechanical stimuli.

Characterization of the endolysosomal system in human chordoma cell lines: is there a role of lysosomes in chemoresistance of this rare bone tumor?

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve "cellular suicide bags". Destabilizing these "suicide bags" might be a promising strategy for the treatment of chordoma.