Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM)

High resolution episcopic microscopy (HREM) produces digital volume data by physically sectioning histologically processed specimens, while capturing images of the subsequently exposed block faces. Our study aims to systematically define the spectrum of typical artefacts inherent to HREM data and to research their effect on the interpretation of the phenotype of wildtype and mutant mouse embryos. A total of 607 (198 wildtypes, 409 mutants) HREM data sets of mouse embryos harvested at embryonic day (E) 14.5 were systematically and comprehensively examined. The specimens had been processed according to essentially identical protocols. Each data set comprised 2000 to 4000 single digital images. Voxel dimensions were 3 × 3 × 3 µm3. Using 3D volume models and virtual resections, we identified a number of characteristic artefacts and grouped them according to their most likely causality. Furthermore, we highlight those that affect the interpretation of embryo data and provide examples for artefacts mimicking tissue defects and structural pathologies. Our results aid in optimizing specimen preparation and data generation, are vital for the correct interpretation of HREM data and allow distinguishing tissue defects and pathologies from harmless artificial alterations. In particular, they enable correct diagnosis of pathologies in mouse embryos serving as models for deciphering the mechanisms of developmental disorders.

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Three-dimensional Sonography in the Assessment of Normal Fetal Anatomy in Late Pregnancy

Donald School Journal of Ultrasound in Obstetrics & Gynecology ◽

10.5005/jp-journals-10009-1145 ◽

2010 ◽

Vol 4 (3) ◽

pp. 217-231

Author(s):

Badreldeen Ahmed ◽

Ulrich Honemeyer

Keyword(s):

Continuous Wave ◽

Power Doppler ◽

Three Dimensional ◽

Late Pregnancy ◽

Research Field ◽

Surface Rendering ◽

Volume Data ◽

Data Set ◽

3D Volume ◽

Color And Power Doppler

Abstract Three-dimensional, multiplanar sonography, using a volume data set acquired with a 3D probe, has revolutionized ultrasonographic imaging and takes sonographers to a new perception of the fetus in 3 dimensions. Real time scanning, until the late nineties only possible in B-mode, can now be performed in 3D with up to 40 frames/sec. Fetal neurology emerged as a new perinatal research field with the 4D visualization of fetal behavior. Doppler ultrasound, diversified and refined from continuous wave and pulsed Doppler to Color – and Power Doppler, when added to 3D sonography, creates fascinating options of noninvasive fetal vascular mapping (sonoangiography) and vascular assessment of placenta. The diagnostic and demonstrative potential of an acquired 3D volume data set can be maxed with the help of postprocessing and rendering software. After storage, the evaluation of fetal 3D data sets can happen without the patient, with the option of specialist consultation, using telemedicine. In the article, the new 3D “modes” like surface rendering, maximum mode, 3D Color and Power Doppler, STIC, volume rendering, and glass body rendering, are described and illustrated in their display of normal fetal anatomy.

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High-Resolution Episcopic Microscopy (HREM): Looking Back on 13 Years of Successful Generation of Digital Volume Data of Organic Material for 3D Visualisation and 3D Display

Applied Sciences ◽

10.3390/app9183826 ◽

2019 ◽

Vol 9 (18) ◽

pp. 3826 ◽

Cited By ~ 1

Author(s):

Stefan H. Geyer ◽

Wolfgang J. Weninger

Keyword(s):

High Resolution ◽

Three Dimensional ◽

3D Analysis ◽

Data Generation ◽

Volume Data ◽

Scientific Publications ◽

3D Visualisation ◽

Broad Variety ◽

Technical Details ◽

Rapid Generation

High-resolution episcopic microscopy (HREM) is an imaging technique that permits the simple and rapid generation of three-dimensional (3D) digital volume data of histologically embedded and physically sectioned specimens. The data can be immediately used for high-detail 3D analysis of a broad variety of organic materials with all modern methods of 3D visualisation and display. Since its first description in 2006, HREM has been adopted as a method for exploring organic specimens in many fields of science, and it has recruited a slowly but steadily growing user community. This review aims to briefly introduce the basic principles of HREM data generation and to provide an overview of scientific publications that have been published in the last 13 years involving HREM imaging. The studies to which we refer describe technical details and specimen-specific protocols, and provide examples of the successful use of HREM in biological, biomedical and medical research. Finally, the limitations, potentials and anticipated further improvements are briefly outlined.

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NanoCT: Visualizing of internal 3D-Structures with Submicrometer Resolution

MRS Proceedings ◽

10.1557/proc-0990-b05-09 ◽

2007 ◽

Vol 990 ◽

Cited By ~ 1

Author(s):

Oliver Brunke ◽

Dirk Neuber ◽

David K. Lehmann

Keyword(s):

High Resolution ◽

Three Dimensional ◽

Dimensional Structure ◽

Volume Data ◽

High Resolution Computed Tomography ◽

Data Set ◽

Minimal Sample ◽

Synthetic Materials ◽

Volume Visualisation ◽

Ct System

ABSTRACTHigh-resolution Computed Tomography (CT) widely expands the spectrum of detectable internal micro-structures. The new nanotom CT system allows the analysis of samples with the exceptional voxel-resolution of less than 0.5 microns per volume pixel (voxel). Thus internal detail related to a variation in material, density or porosity can be visualised and precisely measured. This opens a new dimension of 3D-microanalysis and will partially substitute traditional destructive methods in industrial quality control and research.The nanotom® is the first 180 kV nanoCT system world-wide which is tailored specifically to the highest-resolution applications in material science, micro electronics, geology and biology. Therefore it is particularly suitable for nanoCT-examinations of material samples of any type like synthetic materials, ceramics, composite materials, mineral and organic samples.Several results of high-resolution nanoCT demonstrate the capability to analyse the 3D-microstructure of materials with only minimal sample preparation. For instance, it is possible to image different metal phases in solder joints, the texture of fibres in composites or the volume and distribution of voids in castings.The volume data set is visualised by slices or compiled in a three-dimensional view which can be displayed in various ways. By means of volume visualisation software, the three-dimensional structure of the reconstructed volume can be easily analysed for pores, cracks, and materials density and distribution with the highest magnification and image quality available.By granting the user the ability to navigate the internal structure of an object slice-by-slice with highest resolution in a non-destructive manner, the nanotom creates new possibilities for analysis which have so far been unreachable.

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X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells, tissues, and organs

10.1101/2020.12.18.423480 ◽

2020 ◽

Author(s):

Keith E. Duncan ◽

Kirk J. Czymmek ◽

Ni Jiang ◽

August C. Thies ◽

Christopher N. Topp

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Plant Sample ◽

Volume Data ◽

X Ray ◽

Correlative Imaging ◽

Technical Advances ◽

Plant Level ◽

Light Fluorescence ◽

3D Volume

AbstractCapturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution 3D volumes of intact plant samples from the cell to whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High quality 3D volume data from our enhanced methods facilitate more sophisticated and effective computational segmentation and analyses than have previously been employed for X-ray based imaging. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.

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High-Resolution Episcopic Microscopy (HREM): A Tool for Visualizing Skin Biopsies

Microscopy and Microanalysis ◽

10.1017/s1431927614013063 ◽

2014 ◽

Vol 20 (5) ◽

pp. 1356-1364 ◽

Cited By ~ 13

Author(s):

Stefan H. Geyer ◽

Maria M. Nöhammer ◽

Markus Mathä ◽

Lukas Reissig ◽

Ines E. Tinhofer ◽

...

Keyword(s):

High Resolution ◽

Collagen Fiber ◽

Three Dimensional ◽

Melanocytic Nevus ◽

Fiber Bundles ◽

Structural Level ◽

Data Generation ◽

Volume Data ◽

Skin Biopsies ◽

3D Representations

AbstractWe evaluate the usefulness of digital volume data produced with the high-resolution episcopic microscopy (HREM) method for visualizing the three-dimensional (3D) arrangement of components of human skin, and present protocols designed for processing skin biopsies for HREM data generation. A total of 328 biopsies collected from normally appearing skin and from a melanocytic nevus were processed. Cuboidal data volumes with side lengths of ~2×3×6 mm3 and voxel sizes of 1.07×1.07×1.5 µm3 were produced. HREM data fit ideally for visualizing the epidermis at large, and for producing highly detailed volume and surface-rendered 3D representations of the dermal and hypodermal components at a structural level. The architecture of the collagen fiber bundles and the spatial distribution of nevus cells can be easily visualized with volume-rendering algorithms. We conclude that HREM has great potential to serve as a routine tool for researching and diagnosing skin pathologies.

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Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM)

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd5040058 ◽

2018 ◽

Vol 5 (4) ◽

pp. 58 ◽

Cited By ~ 3

Author(s):

Wolfgang Weninger ◽

Barbara Maurer-Gesek ◽

Lukas Reissig ◽

Fabrice Prin ◽

Robert Wilson ◽

...

Keyword(s):

High Resolution ◽

Cardiovascular System ◽

Human Skin ◽

Developmental Disorders ◽

Mouse Embryos ◽

Model Organisms ◽

Biomedical Model ◽

Cardiovascular Development ◽

Cardiovascular Malformations ◽

Adult Human Skin

The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.

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Algorithms for automated montage synthesis of images from laser-scanning confocal microscopes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100139627 ◽

1995 ◽

Vol 53 ◽

pp. 650-651

Author(s):

D. E. Becker

Keyword(s):

Image Analysis ◽

High Resolution ◽

Laser Scanning ◽

Cell Counting ◽

Wide Area ◽

Data Set ◽

Computational Synthesis ◽

Automated Cell Counting ◽

Partial View ◽

The Individual

An efficient, robust, and widely-applicable technique is presented for computational synthesis of high-resolution, wide-area images of a specimen from a series of overlapping partial views. This technique can also be used to combine the results of various forms of image analysis, such as segmentation, automated cell counting, deblurring, and neuron tracing, to generate representations that are equivalent to processing the large wide-area image, rather than the individual partial views. This can be a first step towards quantitation of the higher-level tissue architecture. The computational approach overcomes mechanical limitations, such as hysterisis and backlash, of microscope stages. It also automates a procedure that is currently done manually. One application is the high-resolution visualization and/or quantitation of large batches of specimens that are much wider than the field of view of the microscope.The automated montage synthesis begins by computing a concise set of landmark points for each partial view. The type of landmarks used can vary greatly depending on the images of interest. In many cases, image analysis performed on each data set can provide useful landmarks. Even when no such “natural” landmarks are available, image processing can often provide useful landmarks.

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High-resolution microscopy of twin boundaries in gold

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100126202 ◽

1987 ◽

Vol 45 ◽

pp. 260-261

Author(s):

William Krakow ◽

David A. Smith

Keyword(s):

High Resolution ◽

Specimen Preparation ◽

Gold Films ◽

Boundary Area ◽

Transmission Electron ◽

Recent Developments ◽

Tilt Boundaries ◽

High Resolution Microscopy ◽

Twist Boundaries

Recent developments in specimen preparation, imaging and image analysis together permit the experimental determination of the atomic structure of certain, simple grain boundaries in metals such as gold. Single crystal, ∼125Å thick, (110) oriented gold films are vapor deposited onto ∼3000Å of epitaxial silver on (110) oriented cut and polished rock salt substrates. Bicrystal gold films are then made by first removing the silver coated substrate and placing in contact two suitably misoriented pieces of the gold film on a gold grid. Controlled heating in a hot stage first produces twist boundaries which then migrate, so reducing the grain boundary area, to give mixed boundaries and finally tilt boundaries perpendicular to the foil. These specimens are well suited to investigation by high resolution transmission electron microscopy.

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High-resolution observation of sintered alumina (Al2O3) using the specimen-heated and electron-beam-induced conductivity method in a UHR-SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100172966 ◽

1994 ◽

Vol 52 ◽

pp. 1046-1047

Author(s):

K. Ogura ◽

T. Suzuki ◽

C. Nielsen

Keyword(s):

Electron Beam ◽

High Resolution ◽

Specimen Preparation ◽

Conductivity Method ◽

Fine Grain ◽

Grain Structures ◽

Resolution Observation ◽

Electron Microscopes ◽

Scanning Electron Microscopes ◽

Charging Effect

In spite of the complicated specimen preparation, Transmission Electron Microscopes (TEM) have traditionally been used for the investigation of the fine grain structures of sintered ceramics. Scanning Electron Microscopes (SEM) have not been used much for the same purpose as TEM because of poor results caused by the specimen charging effect, and also the lack of sufficient resolution. Here, we are presenting a successful result of high resolution imaging of sintered alumina (pure Al2O3) using the Specimen Heated and Electron Beam Induced Conductivity (SHEBIC) method, which we recently reported, in an ultrahigh resolution SEM (UHR-SEM). The JSM-6000F, equipped with a Field Emission Gun (FEG) and an in-lens specimen position, was used for this application.After sintered Al2O3 was sliced into a piece approximately 0.5 mm in thickness, one side was mechanically polished to get a shiny plane for the observation. When the observation was started at 20 kV, an enormous charging effect occured, and it was impossible to obtain a clear Secondary Electron (SE) image (Fig.1).

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