Multi-Fluorescence High-Resolution Episcopic Microscopy (MF-HREM) for Three-Dimensional Imaging of Adult Murine Organs

Walsh, Claire; Holroyd, Natalie; Finnerty, Eoin; Ryan, Sean G.; Sweeney, Paul W.; Shipley, Rebecca J.; Walker‐Samuel, Simon

doi:10.1101/2020.04.03.023978

Cited by 5 publications

(6 citation statements)

References 81 publications

(176 reference statements)

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“…Although the first publications included examples for visualizing LacZ-stained tissues or whole-mount in situ hybridization, this is still experimental [20,70]. The use of other embedding techniques to visualize fluorescent signals is also opening new fields of applications for Histo3D that are very promising [71]. In this context, the use of the Nanocut rotary microtome with Histo3D will be a powerful technique to process very small samples as it is able to use sections as thin as 1 µm or even less.…”

Section: Discussionmentioning

confidence: 99%

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System

et al. 2021

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“…Other quantification methods began appearing in papers from 2017 onwards. These include structure tensor analysis for calculating myocyte and neuron orientation [6,19,20], skeletonization algorithms for producing a graphical representation of vasculature [6,21], and measures of asymmetry [22].…”

Section: Trends In Methods Of Quantificationmentioning

confidence: 99%

“…Automated and semi-automated methods were less widely used: intensity thresholding was employed in six papers, while vasculature was segmented via Frangi filtering in two papers [23,24]. Some papers employed as specialized methods such as the MOST tracing algorithm in Vaa3D for neuron tracing [6,25] and the Vascular Modelling Toolkit (VMTK) for vessel segmentation [26]. A wide range of software was used to facilitate episcopic image analysis.…”

Section: Methods Of Image Annotationmentioning

confidence: 99%

“…Episcopic Fluorescence Image Capture (EFIC) is a variant of HREM in which samples are embedded in non-fluorescent resin and contrast is provided solely by the native autofluorescence of the tissue [5]. More recently MF-HREM was developed to enable imaging of multiple targeted fluorescent stains in a similar manner to other 3D optical imaging modalities such as light sheet or serial two-photon microscopy [6].…”

Section: Background On Episcopic Imaging Techniquesmentioning

confidence: 99%

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Quantitative Image Processing for Three-Dimensional Episcopic Images of Biological Structures: Current State and Future Direction

Holroyd¹,

Walsh²,

Gourmet³

et al. 2023

Preprint

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Episcopic imaging using techniques such as High Resolution Episcopic Microscopy (HREM) and its variants, allows biological samples to be visualized in three dimensional over a large field of view. Quantitative analysis of these images is undertaken using a range of methods. In this systematic review, we look at trends in quantitative analysis of episcopic images and discuss avenues for further research. Papers published between 2011 and 2022 were analyzed for details about quantitative analysis approaches, methods of image annotation and choice of image processing software. It is shown that quantitative processing is becoming more common in episcopic microscopy and that manual annotation is the predominant method of image analysis. Our meta-analysis highlights where tools and methods still require development in this field, and we discuss what this means for the future of quantitative episcopic imaging, as well as how annotation and quantification may be automated and standardized across the field.

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“…The data that support the findings of this study are openly available in Image Data Resource (IDR) at https://doi.org/10.17867/10000143. [62]…”

Section: Supporting Informationmentioning

confidence: 99%

Multifluorescence High‐Resolution Episcopic Microscopy for 3D Imaging of Adult Murine Organs

Walsh

Holroyd

Finnerty

et al. 2021

Advanced Photonics Research

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3D microscopy of large biological samples (>0.5 cm 3 ) is transforming biological research. Many existing techniques require trade-offs between image resolution, sample size, and method complexity. A simple robust instrument with the potential to conduct large-volume 3D imaging currently exists in the form of the optical high-resolution episcopic microscopy (HREM). However, the development of the instrument to date is limited to single-fluorescent wavelength imaging with nonspecific eosin staining. Herein, developments to realize the potential of the HREM to become multifluorescent high-resolution episcopic microscopy (MF-HREM) are presented. MF-HREM is a serial-sectioning and block-facing wide-field fluorescence imaging technique, which does not require tissue clearing or optical sectioning. Multiple developments are detailed in sample preparation and image postprocessing to enable multiple specific stains in large samples and show how these enable segmentation and quantification of the data. The application of MF-HREM is demonstrated in a variety of biological contexts: 3D imaging of whole tumor vascular networks and tumor cell invasion in xenograft tumors up to 7.5 mm 3 at resolutions of 2.75 μm, quantification of glomeruli volume in the adult mouse kidney, and quantification of vascular networks and white-matter track orientation in adult mouse brain.

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Multi-Fluorescence High-Resolution Episcopic Microscopy (MF-HREM) for Three-Dimensional Imaging of Adult Murine Organs

Cited by 5 publications

References 81 publications

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System

Quantitative Image Processing for Three-Dimensional Episcopic Images of Biological Structures: Current State and Future Direction

Multifluorescence High‐Resolution Episcopic Microscopy for 3D Imaging of Adult Murine Organs

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