HREM is based on unspecific contrasting and provides highly detailed information of the overall morphology of various biologic specimens. Combining this detailed structural information with the exact localisation of specifically labelled structures, gene expression and gene product patterns is a highly beneficial tool to unravel basic developmental and pathologic mechanisms. Thus, attempts to detect specifically stained structures were undertaken from the very first beginnings of HREM.

Due to methodical constraints, the visualisation of specific signals has to be performed after wholemount staining prior to the embedding of the specimen, which is challenging for large samples and hard-to-penetrate tissues. Nevertheless, it has been achieved following wholemount in situ hybridisation, wholemount immuno-staining and lacZ staining utilising two of the multiband filter sets during HREM data generation [27]. More recently, multifluorescence HREM (MF-HREM) utilising fluorescent dyes and opaque resin has been introduced [28].

The production and screening of mutant embryos in large-scale phenotyping studies is expensive and time consuming. Imaging pipelines that facilitate rapid pre-selection of samples or definition of volumes of interest at lower resolution, which is then followed by detailed validation at higher resolution, could provide an efficient alternative.

Both micro-MRI and HREM have been established as routine tools for analysing phenotypes of genetically altered mouse embryos. While micro-MRI offers the advantage of being able to simultaneously scan multiple embryos at the organ and organ-system level in one run, HREM offers superior near-histological image quality. The combination of both techniques allows for identifying embryos of interest that show an ambiguous phenotype or a phenotype hinting at additional abnormalities with micro-MRI. These embryos are then subjected to HREM for further analysis [26].

This also allowed for a better management of the large amounts of data typically generated during acquisition. A combination of both modalities offered a continuous high-throughput imaging pipeline.

Chick embryos are a valuable model organism both for developmental and cancer biology. They can be used to study embryonic morphogenesis and develop experimental surgical techniques. They are also especially useful when studying tumour genesis. To establish imaging pipelines combining in vivo observations and high resolution, various modalities must be combined. OCT and PAT are techniques that have previously been successfully used in vivo [29] and in utero [30], respectively. A pipeline integrating OCT/PAT has been established for chick embryos at several developmental stages. Following these modalities, HREM then offered high-resolution insights into regions of interest [31].

Overall, this pipeline has the potential to facilitate the analysis and monitoring of tumour genesis, organogenesis, and the development of vasculature in embryos.

An area of interest in developmental biology is the establishment of the left-right body axis and its underlying molecular mechanisms during early development. The heart as an asymmetric organ is an important model for left-right morphogenesis, and laterality defects are the cause for many complex congenital heart defects. To track embryonic development, in vivo imaging techniques—such as small-animal US and micro-CT—are of special interest to those research questions, coupled with high-resolution analysis methods that can reveal morphological abnormalities in detail.

In the study of Desgrange et al. [32], murine embryos at different stages of development were imaged, with a special focus on the development of the heart loop. Using micro-US, embryos were visualised in vivo at E9.5, and their positions in the uterine horns of the pregnant mouse determined. This was followed by micro-CT and HREM after sacrificing the embryos at E18.5. Because of the non-invasiveness of micro-CT, it was possible to check the situs of the visceral organs within the embryo without dissection. Afterwards, organs of interest, such as the heart, were excised and prepared for visualisation with HREM. Structural abnormalities were then determined at higher resolution.

This pipeline is not restricted to analyse laterality defects and can be easily modified to focus on other organs and developmental stages.

Tumour vasculature plays a major role for tumour progression and dissemination, highlighting the importance of vascular visualisation. The application of CMI pipelines enables the depiction of multiple vascular parameters of the same tumour across scales and penetration depths.

As part of a proof-of-principle study, a novel CMI pipeline was established to characterise aberrant tumour vasculature in a murine orthotopic melanoma model. This CMI approach by Zopf et al. [15] incorporated modalities such as MRI, PET, CT, OCT, HREM, and HP (Figure 3). This novel platform explored the feasibility of combining these technologies using an extensive image processing pipeline. In this workflow, HREM offered the highest resolution of about 3 µm. Two-dimensional HREM images offered near-histologic resolution, and the structures were further resolved and confirmed by post-HREM HP. For post-HREM HP, JB-4 resin sections were collected during HREM imaging, stained, and additionally evaluated to validate the intratumoral vessels that were segmented based on the HREM images. Correlative registration was achieved between H & E-stained JB-4 sections (collected during HREM imaging) and 2D HREM images by using the morphologic landmarks of skin, subcutaneous vessels, nerve bundles, and mammary ducts. Importantly, blood vessels did not need to be perfused or selectively contrasted for 3D imaging, which is why HREM, in addition to visualising capillaries inside and outside the tumour, also allowed one to visualise capsules, blindly terminating or beginning vessels, and short collaterals inside the tumour and necrotic tissue.

The combination of in vivo imaging with ex vivo biological microscopy enables the visualisation of both the whole organism and single structures within. With the aim of characterising vascular lesions in a genetically engineered mouse model, a novel multimodal workflow was established as described in Keuenhof et al. [16] by combining micro-MRI and micro-CT with HREM to identify and study vascular abnormalities using a targeted knockout mouse model (Figure 4). The pipeline allowed the study of the same VOI for each modality at different length scales, from an overview at the organ level (micro-MRI, micro-CT) to micrometre resolution (HREM).

For an overview at the macroscopic scale of the entire mouse vasculature and the detection of suspected vascular lesions, micro-MRI was used with a lateral resolution of about 100 × 50 µm². With contrast-enhanced ex vivo micro-CT, blood vessels in the region of interest (identified as the left hindlimb by in vivo imaging) were examined in further detail at an isotropic resolution of about 15 µm which revealed a vessel suspected of blockage. Subsequently, the limb of interest was isolated en bloc including the vessel of interest, for further characterisation of the site of suspected occlusion at a resolution of 3 µm by HREM and HP. HREM suggested a narrowing of the vessel lumen and evoked suspicion of intimal hyperplasia in the affected blood vessel segment within the VOI (Figure 4). Only the inclusion of HREM in this pipeline allowed the detection of this narrowing of the vessel lumen and vascular intimal hyperplasia within the VOIs identified by CT and MRI [16].