Biology subjects

Svetlove, A.

Publications and source records attributed to Svetlove, A..

5 recordsLinked to original sources

bioRxiv2026

Sample preparation optimisation for array tomography volume EM of entire organisms: a case study for marine larvae

Serial-sectioning electron microscopy allows the exploration of the 3D ultrastructure of cells, tissues or whole organisms. Sample preparation is a critical step for this technique, which determines the quality of downstream imaging and analysis. Here we optimize a high-pressure freezing sample-preparation workflow for larvae of the marine annelid Platynereis dumerilii. We tested several fixation and staining methods and screened a large number of specimens by synchrotron X-ray imaging to evaluate specimen integrity and contrast. We further evaluated selected blocks by array tomography SEM to select the best performing protocols. Our protocols and robust workflow could be adapted to similar marine planktonic organisms including ciliated invertebrate larvae.

developmental biology↗

bioRxiv2025

Critical point drying of brain tissue for X-ray phase contrast imaging

X-ray phase contrast tomography is emerging as a powerful method for imaging large volumes of brain tissue at sub-cellular resolution. However, current sample preparation methods are largely inherited from visible light or electron microscopy workflows and hence are not optimised to exploit the full potential of X-ray contrast mechanisms. Here we propose to replace interstitial material by air to enhance X-ray phase contrast of the ultrastructural features. We used critical point drying (CPD) of heavy metal-stained mouse brain tissue to produce mechanically stable samples with preserved ultrastructure and enhanced refractive index boundaries, a nanofoam-like material that remains compatible with follow-up conventional resin embedding. Using two complementary synchrotron-based setups, a high-throughput microtomography beamline (P14, DESY) and a nanoscale holographic tomography beamline (ID16A, ESRF), we found that CPD samples consistently showed 2-4x stronger phase-shift signal than conventional resin-embedded tissue. The contrast gain remained consistent across samples, imaging conditions, and beamlines. Our results suggest that CPD offers a versatile route for preparing tissue for subcellular and ultrastructural-resolution X-ray imaging. It retains structural detail while improving signal, and is compatible with other processing procedures like femtosecond laser milling or electron microscopy, paving the path for biological tissue imaging beyond the mm3 scale.

neuroscience↗

bioRxiv2024

Vapor-based Fixation of Pulmonary Tissue in its Physiological State: A Novel Approach to Histological Validation of Ultra High Resolution Phase Contrast CT in Human Sized Lungs

Lung diseases continue to present a major burden on public health. Therefore, improving the process of diagnosis by the development of novel imaging techniques is of great importance. In this perspective, phase sensitive CT imaging techniques such as propagation based imaging (PBI) might play an important role as they allow increasing the spatial resolution at very low x-ray dose levels that are comparable to clinical CT. However, the development of such methods is not only hindered by technological problems but also by the lack of precise validation strategies. We adapted formaldehyde (FA) vapor fixation to demonstrate that fresh porcine lungs that have been investigated by PBI can be fixed in their physiological shape and studied by multi-scale microCT imaging as well as classical histology. In addition, we show that FA vapor fixed pig lungs can be scanned by PBI without visible deterioration of image quality compared to fresh tissue. This opens the possibility of fixing and storing, for instance, human lung tissue before performing a PBI experiment, which in turn allows to study pathological changes in human lungs without questioning the translate-ability of findings in pig lung. The setup can be used by any interested researchers.

pathology↗

bioRxiv2024

Integrative Imaging of Lung Micro Structure: Amplifying Classical Histology by Paraffin Block μCT and same-slide Scanning Electron Microscopy

Classical histopathology of formalin fixed and paraffin embedded (FFPE) tissue using light microscopy (LM) remains the undisputed gold standard in biomedical microstructural lung tissue analysis. To extend this method, we developed an integrative imaging and processing pipeline which adds 3D context and screening capabilities by micro-CT (CT) imaging of the entire paraffin block and adds ultrastructural information by correlative same-slide scanning electron microscopy (SEM). The different modalities are integrated by elastic registration to provide hybrid image datasets. Without compromising standard light microscopic readout, we overcome the limitations of conventional histology by combining and integrating several imaging modalities. The biochemical information contained in histological and immunohistological tissue staining is embedded into the 3D tissue configuration and is amplified by adding ultrastructural visualization of features of interest. By combining CT and conventional histological processing, specimens can be screened, and specifically preselected areas of interest can be targeted in the subsequent sectioning process. While most of the CT data shown in the manuscript was acquired at a Synchrotron, we further demonstrate that our workflow can also by applied using X-ray microscopy.

pathology↗

bioRxiv2023

High Throughput Tomography (HiTT) on EMBL Beamline P14 on PETRA III

Here we present High-Throughput Tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany. We use a high photon flux undulator beamline to perform tomographic phase contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beamtime shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. We demonstrate the successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin embedded preparations. Furthermore, we demonstrate the feasibility of HiTT to be used as a targeting tool for volume electron microscopy (vEM), as well as using HiTT to study plant morphology. We also show how the high throughput nature of the work has allowed large numbers of "identical" samples to be imaged to enable statistically relevant sample volumes to be studied. SynopsisWe present HiTT - high throughput tomography - a propagation based phase contrast X-ray imaging technique which can visualise 1 mm3 biological samples of various types at high resolution. The 3D reconstructions of the imaged volumes are calculated automatically once data collection is complete. The entire process from pressing start on data collection to viewing the final data takes less than 3 minutes. This speed in combination with the use of the automated sample changer to exchange the samples truly enables high throughput X-ray imaging for the first time.

bioengineering↗