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Biomedicines
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Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution...
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Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging

A special issue of Biomedicines (ISSN 2227-9059). This special issue belongs to the section "Biomedical Engineering and Materials".

Deadline for manuscript submissions: closed (31 October 2021) | Viewed by 16117

Special Issue Editors

Prof. Dr. Wolfgang J. Weninger

E-Mail Website
Guest Editor
Division of Anatomy & Medical Imaging Cluster, Medical University of Vienna, Vienna, Austria
Interests: anatomy; episcopic 3D imaging; morphology; human; embryogenesis
Special Issues, Collections and Topics in MDPI journals
Dr. Stefan H. Geyer

E-Mail Website
Guest Editor
Division of Anatomy, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria
Interests: imaging; morphogenesis; histology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

HREM is a digital volume data generation technique, which offers near histological detail in virtual 3D data of whole embryos of biomedical model organisms and small tissue samples. Introduced over 10 years ago, the HREM user community is steadily growing and employs the method for phenotyping genetically engineered mouse embryos, biomedically challenged chick embryos and embryos of several other model organisms, as well as normal and pathological tissue samples of adult biomedical models and humans. Quite recently HREM also found its way into multimodal imaging pipelines providing holistic visualization of normal and pathologic morphology and physiology of organisms at all levels of resolution. We invite authors to submit review articles focusing on applying HREM in the various fields of biomedical research.

Prof. Dr. Wolfgang J. Weninger
Dr. Stefan H. Geyer
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomedicines is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • episcopic imaging
  • phenotyping
  • bioimaging
  • high-resolution episcopic microscopy

Published Papers (6 papers)

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Editorial

Jump to: Research, Review

3 pages, 178 KiB  
Editorial
Visualizing 3D Embryo and Tissue Morphology—A Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging
by Stefan H. Geyer and Wolfgang J. Weninger
Biomedicines 2022, 10(5), 1123; https://doi.org/10.3390/biomedicines10051123 - 12 May 2022
Cited by 1 | Viewed by 2766
Abstract
High-resolution episcopic microscopy (HREM) [...] Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)

Research

Jump to: Editorial, Review

24 pages, 15095 KiB  
Article
HREM, RNAseq and Cell Cycle Analyses Reveal the Role of the G2/M-Regulatory Protein, WEE1, on the Survivability of Chicken Embryos during Diapause
by Narayan Pokhrel, Olga Genin, Dalit Sela-Donenfeld and Yuval Cinnamon
Biomedicines 2022, 10(4), 779; https://doi.org/10.3390/biomedicines10040779 - 27 Mar 2022
Cited by 4 | Viewed by 2238
Abstract
Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular [...] Read more.
Avian blastoderm can enter into diapause when kept at low temperatures and successfully resume development (SRD) when re-incubated in body temperature. These abilities, which are largely affected by the temperature and duration of the diapause, are poorly understood at the cellular and molecular level. To determine how temperature affects embryonic morphology during diapause, high-resolution episcopic microscopy (HREM) analysis was utilized. While blastoderms diapausing at 12 °C for 28 days presented typical cytoarchitecture, similar to non-diapaused embryos, at 18 °C, much thicker blastoderms with higher cell number were observed. RNAseq was conducted to discover the genes underlying these phenotypes, revealing differentially expressed cell cycle regulatory genes. Among them, WEE1, a negative regulator of G2/M transition, was highly expressed at 12 °C compared to 18 °C. This finding suggested that cells at 12 °C are arrested at the G2/M phase, as supported by bromodeoxyuridine incorporation (BrdU) assay and phospho-histone H3 (pH 3) immunostaining. Inhibition of WEE1 during diapause at 12 °C resulted in cell cycle progression beyond the G2/M and augmented tissue volume, resembling the morphology of 18 °C-diapaused embryos. These findings suggest that diapause at low temperatures leads to WEE1 upregulation, which arrests the cell cycle at the G2/M phase, promoting the perseverance of embryonic cytoarchitecture and future SRD. In contrast, WEE1 is not upregulated during diapause at higher temperature, leading to continuous proliferation and maladaptive morphology associated with poor survivability. Combining HREM-based analysis with RNAseq and molecular manipulations, we present a novel mechanism that regulates the ability of diapaused avian embryos to maintain their cytoarchitecture via cell cycle arrest, which enables their SRD. Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)
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18 pages, 4321 KiB  
Article
Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM)
by Lukas F. Reissig, Stefan H. Geyer, Julia Rose, Fabrice Prin, Robert Wilson, Dorota Szumska, Antonella Galli, Catherine Tudor, Jacqueline K. White, Tim J. Mohun and Wolfgang J. Weninger
Biomedicines 2021, 9(11), 1711; https://doi.org/10.3390/biomedicines9111711 - 18 Nov 2021
Cited by 5 | Viewed by 1435
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)
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18 pages, 4478 KiB  
Article
High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System
by Olivia Wendling, Didier Hentsch, Hugues Jacobs, Nicolas Lemercier, Serge Taubert, Fabien Pertuy, Jean-Luc Vonesch, Tania Sorg, Michela Di Michele, Laurent Le Cam, Thomas Rosahl, Ester Carballo-Jane, Mindy Liu, James Mu, Manuel Mark and Yann Herault
Biomedicines 2021, 9(7), 767; https://doi.org/10.3390/biomedicines9070767 - 1 Jul 2021
Cited by 7 | Viewed by 3158
Abstract
3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be [...] Read more.
3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice. Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)
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18 pages, 3333 KiB  
Article
Pathogenesis of Anorectal Malformations in Retinoic Acid Receptor Knockout Mice Studied by HREM
by Manuel Mark, Marius Teletin, Olivia Wendling, Jean-Luc Vonesch, Betty Féret, Yann Hérault and Norbert B. Ghyselinck
Biomedicines 2021, 9(7), 742; https://doi.org/10.3390/biomedicines9070742 - 28 Jun 2021
Cited by 6 | Viewed by 2633
Abstract
Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital [...] Read more.
Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses. Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)
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Review

Jump to: Editorial, Research

18 pages, 4612 KiB  
Review
High-Resolution Episcopic Microscopy (HREM) in Multimodal Imaging Approaches
by Katharina S. Keuenhof, Anoop Kavirayani, Susanne Reier, Stefan H. Geyer, Wolfgang J. Weninger and Andreas Walter
Biomedicines 2021, 9(12), 1918; https://doi.org/10.3390/biomedicines9121918 - 15 Dec 2021
Cited by 7 | Viewed by 2831
Abstract
High-resolution episcopic microscopy (HREM) is a three-dimensional (3D) episcopic imaging modality based on the acquisition of two-dimensional (2D) images from the cut surface of a block of tissue embedded in resin. Such images, acquired serially through the entire length/depth of the tissue block, [...] Read more.
High-resolution episcopic microscopy (HREM) is a three-dimensional (3D) episcopic imaging modality based on the acquisition of two-dimensional (2D) images from the cut surface of a block of tissue embedded in resin. Such images, acquired serially through the entire length/depth of the tissue block, are aligned and stacked for 3D reconstruction. HREM has proven to be specifically advantageous when integrated in correlative multimodal imaging (CMI) pipelines. CMI creates a composite and zoomable view of exactly the same specimen and region of interest by (sequentially) correlating two or more modalities. CMI combines complementary modalities to gain holistic structural, functional, and chemical information of the entire sample and place molecular details into their overall spatiotemporal multiscale context. HREM has an advantage over in vivo 3D imaging techniques on account of better histomorphologic resolution while simultaneously providing volume data. HREM also has certain advantages over ex vivo light microscopy modalities. The latter can provide better cellular resolution but usually covers a limited area or volume of tissue, with limited 3D structural context. HREM has predominantly filled a niche in the phenotyping of embryos and characterisation of anatomic developmental abnormalities in various species. Under the umbrella of CMI, when combined with histopathology in a mutually complementary manner, HREM could find wider application in additional nonclinical and translational areas. HREM, being a modified histology technique, could also be incorporated into specialised preclinical pathology workflows. This review will highlight HREM as a versatile imaging platform in CMI approaches and present its benefits and limitations. Full article
(This article belongs to the Special Issue Visualizing 3D Embryo and Tissue Morphology—a Decade of Using High-Resolution Episcopic Microscopy (HREM) in Biomedical Imaging)
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