Carl Zeiss AG

Purpose: To establish normative data for macular thickness, macular volume and peripapillary retinal nerve fibre layer (RNFL) thickness using Spectralis® spectral-domain optical coherence tomography (SD-OCT) in healthy German children and adolescents and investigate influencing factors. Methods: The cross-sectional study included the right eye of 695 children with at least one complete retinal OCT scan. As part of the LIFE Child study, the children underwent an ophthalmological examination including axial length (AL), spherical equivalent (SE) and OCT measurements. Various questionnaires were answered by the children or their parents to identify media use or outdoor time. Multiple linear regression models were used to investigate the potential influencing factors. Results: A total of 342 boys and 353 girls with an average age (SD) of 12.91 (3.29) years participated. The mean AL (SD) was 23.20 (0.86) mm. The mean macular thickness (SD) was 320.53 (12.29) μm and the mean RNFL thickness (SD) was 102.88 (8.79) μm. Statistical analysis revealed a significant correlation between average macular thickness and age (p < 0.001, β = 0.77) as well as AL (p < 0.001, β = -4.06). In addition, boys had thicker maculae (p < 0.001, β = 5.36). The RNFL thickness showed no significant correlation with children's age (p > 0.05), but with AL (p = 0.002, β = -2.15), birth weight (p = 0.02, β = 0.003) and a gender-specific effect of the body mass index standard deviation score for male participants (p = 0.02, β = 1.93). Conclusion: This study provides normative data and correlations between macular and RNFL thickness in healthy German children. Especially age, gender and AL must be taken into account when evaluating quantitative OCT measurements to classify them as normal.

A seismic investigation on Saturn's moon Enceladus could determine the thickness of the ice shell, along with variations from the mean thickness, by recovering phase and group velocities, and through the frequency content of surface waves. Here, we model the Enceladus ice shell with uniform thicknesses of 5 km, 20 km, and 40 km, as well as with ice topography ranging from 5-40 km. We investigate several approaches for recovering the mean ice shell thickness. We show that surface wave dispersions could be used to determine the mean ice shell thickness. Flexural waves in the ice only occur if the shell is thinner than a critical value < 20 km. Rayleigh waves dominate only in thicker ice shells. The frequency content of Crary waves depends on the ice shell thickness.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Crossing the blood-brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and end-point in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (ECs) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by crosstalk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it.

Electron-stimulated etching of surfaces functionalized by remote plasma is a flexible and novel approach for material removal. In comparison with plasma dry etching, which uses the ion-neutral synergistic effect to control material etching, electron beam-induced etching (EBIE) uses an electron-neutral synergistic effect. This approach appears promising for the reduction of plasma-induced damage (PID), including atomic displacement and lateral straggling, along with the potential for greater control and lateral resolution. One challenge for EBIE is the limited selection of chemical precursor molecules that can be used to produce functionalized materials suitable for etching under electron beam irradiation. In this work, we studied a new experimental approach that utilizes a remote plasma source to functionalize substrate surfaces in conjunction with electron beam irradiation by an electron flood gun. Etching rates (ERs) of SiO 2 , Si 3 N 4 , and poly-Si are reported in a broad survey of processing conditions. The parametric dependence of the ER of these Si-based materials on the operating parameters of the flood gun and the remote plasma source is evaluated. We also identified the processing parameters that enable the realization of material selective removal, i.e., the etching selectivity of Si 3 N 4 over SiO 2 and poly-Si over SiO 2 . Additionally, surface characterization of etched materials is used to clarify the effects of the co-introduction of particle fluxes from the remote plasma and flood gun sources on surface chemistry.

In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope’s field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2–6. However, the networks’ architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs. A population of highly interconnected cells in glioblastoma makes these tumours resistant to general damage but vulnerable to targeted disruption of this small fraction of cells and their rhythmic Ca2+ oscillations.

Electron-based surface activation of surfaces functionalized by remote plasma appears like a flexible and novel approach to atomic scale etching and deposition. Relative to plasma-based dry etching that uses ion bombardment of a substrate to achieve controlled material removal, electron beam-induced etching (EBIE) is expected to reduce surface damage, including atom displacement, surface roughness, and undesired material removal. One of the issues with EBIE is the limited number of chemical precursors that can be used to functionalize material surfaces. In this work, we demonstrate a new configuration that was designed to leverage flexible surface functionalization using a remote plasma source, and, by combining with electron beam bombardment to remove the chemically reacted surface layer through plasma-assisted electron beam-induced etching, achieve highly controlled etching. This article describes the experimental configuration used for this demonstration that consists of a remote plasma source and an electron flood gun for enabling electron beam-induced etching of SiO 2 with Ar/CF 4 /O 2 precursors. We evaluated the parametric dependence of SiO 2 etching rate on processing parameters of the flood gun, including electron energy and emission current, and of the remote plasma source, including radiofrequency source power and flow rate of CF 4 /O 2 , respectively. Additionally, two prototypical processing cases were demonstrated by temporally combining or separating remote plasma treatment and electron beam irradiation. The results validate the performance of this approach for etching applications, including photomask repair and atomic layer etching of SiO 2 . Surface characterization results that provide mechanistic insights into these processes are also presented and discussed.

For an increasing number of applications, the quality and the stability of manufacturing processes can be determined via image and video-image data analysis and new techniques are required to extract and synthesize the relevant information content enclosed in big sensor data to draw conclusions about the process and the final part quality. This paper focuses on video image data where the phenomena under study is captured by a point process whose spatial signature is of interest. A novel approach is proposed which combines spatial data modeling via Ripley’s K-function with Functional Analysis of Variance (FANOVA), i.e., Analysis of Variance on Functional data. The K-function allows to synthesize the spatial pattern information in a function while preserving the capability to capture changes in the process behavior. The method is applicable to quantities and phenomena that can be represented as clusters, or clouds, of spatial points evolving over time. In our case, the motivating case study regards the analysis of spatter ejections caused by the laser-material interaction in Additive Manufacturing via Laser Powder Bed Fusion (L-PBF). The spatial spread of spatters, captured in the form of point particles through in-situ high speed machine vision, can be used as a proxy to select the best conditions to avoid defects (pores) in the manufactured part. The proposed approach is shown to be not only an efficient way to translate the high-dimensional video image data into a lower dimensional format (the K-function curves), but also more effective than benchmark methods in detecting departures from a stable and in-control state.

The correlative imaging workflow is a method of combining information and data across modes (e.g., SEM, X‐ray CT, FIB‐SEM), scales (cm‐to‐nm) and dimensions (2D‐3D‐4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multi‐layered samples) are identified from initial exploratory non‐destructive 3D tomographic imaging (e.g. X‐ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm‐to‐nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broadbeam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF‐SEM), and ion milling and laser approaches such as FIB‐SEM, Xe plasma FIB‐SEM, and femtosecond laser/laser FIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non‐correlative workflows. This article is protected by copyright. All rights reserved

Single cell analysis is crucial for elucidating cellular diversity and heterogeneity as well as for medical diagnostics operating at the ultimate detection limit. Although superbly sensitive biosensors have been developed using the strongly enhanced evanescent fields provided by optical microcavities, real-time quantification of intracellular molecules remains challenging due to the extreme low quantity and limitations of the current techniques. Here, we introduce an active-mode optical microcavity sensing stage with enhanced sensitivity that operates via Förster resonant energy transferring (FRET) mechanism. The mutual effects of optical microcavity and FRET greatly enhances the sensing performance by four orders of magnitude compared to pure Whispering gallery mode (WGM) microcavity sensing system. We demonstrate distinct sensing mechanism of FRET-WGM from pure WGM. Predicted lasing wavelengths of both donor and acceptor by theoretical calculations are in perfect agreement with the experimental data. The proposed sensor enables quantitative molecular analysis at single cell resolution, and real-time monitoring of intracellular molecules over extended periods while maintaining the cell viability. By achieving high sensitivity at single cell level, our approach provides a path toward FRET-enhanced real-time quantitative analysis of intracellular molecules.

Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.