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

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 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.

Detailed characterizations of cranial nerve anatomy in E14.5 mouse embryos/fetuses and their use as reference for diagnosing subtle, but potentially lethal malformations in mutants

“…The correctness of the interpretation was backed by comparisons with histological images ( Kaufman, 1992 ). In addition, HREM data have limitations common to all histological imaging approaches ( Reissig et al, 2021a ). The most significant is that the specimens have to be dehydrated, infiltrated and embedded in resin.…”

“…Third: Similar to adults, embryos/fetuses show an immensely broad spectrum of anatomic variation in topology, morphology and volume of tissues and organs, respectively their components. Hence, identifying anatomic variations and bordering them from pathologies and artefacts is far from being trivial ( Reissig et al, 2021a ). The only reasonable solution to cope with this problem is to compare the phenotype of each mutant with the phenotypes of a sufficiently large number of genetically normal individuals of identic developmental progress.…”

Mouse embryo phenotyping using X-ray microCT

“…Other imaging modalities similarly suffer from artefacts by specimen processing. In HREM, shrinkage is caused by dehydration and JB4 resin embedding but quantified data are missing (Reissig et al, 2021). In LSM/SPIM, clearing may cause massive shrinkage, the extent depending on the type of agent (BABB, Spalteholz fluid, 3DISCO; Miller et al, 2005;Buytaert et al, 2014;Azaripour et al, 2016;Vigouroux et al, 2017;Vulders et al, 2021).…”

“…Frontiers in Cell and Developmental Biology frontiersin.org 13 commercialized by a single vendor (Reissig et al, 2021;Walsh et al, 2021). To date, the availability of HREM to researchers is limited and HREM is less frequently used than microCT.…”