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

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 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.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway

“…For HREM analysis, embryos were dehydrated and embedded in methacrylate resin (JB-4, Polysciences) containing eosin and acridine orange. After polymerisation and hardening, the resin blocks were used for HREM data generation [7,8]. Section thickness was set at 5 μm (E9.5, E10.5, E11.5, and E12.5) and 7 μm (E14.5).…”

Quantitative Neuroanatomical Phenotyping of the Embryonic Mouse Brain

“…It is worthwhile to mention that the protocols presented here can be adapted to cryosections using a cryostat for sectioning of thicker slices of about 30 to 40 µm. While these standard 2D histological practices can provide cost‐effective and relatively straightforward answers when it comes to quantifying NAPs, high‐resolution 3D techniques are becoming increasingly accessible for embryonic brain exploration (Wendling et al., 2021) but are typically low throughput and require important informatic resources to acquire, store, and analyze large datasets (Pan et al., 2016; Zhong et al., 2021). About 80% of congenital neurodevelopmental anomalies are of genetic etiology, and our increasingly sophisticated ability to phenotype the embryonic mouse brain will continue to help in gene discovery in mice, which remains crucial to progress in the postgenomic era (Collins, Mikhaleva, et al., 2019).…”

E4f1 Coordinates Pyruvate Metabolism and the Activity of the Elongator Complex to Ensure Translation Fidelity During Brain Development

“…Fixed samples were gradually dehydrated in an increasing series of ethanol concentrations and were embedded in a methacrylate resin (JB-4 kit, Polysciences, Warrington, PA) containing eosin and acridin orange as contrasts agents as previously described (Mohun and Weninger, 2012) (https://dmdd.org.uk/hrem/). The resin blocks were sectioned on the Histo3D system to generate data by repeated removal of 7 µm thick sections (Wendling et al, 2021). Resulting HREM data with a voxel size of 8 X 8 X 7 µm 3 (E14.5 whole embryos) or 6.5 X 6.5 X 7 µm 3 (E16.5 heads) were generated from approximately 1000 or 800 aligned images, respectively.…”