*2.2. HREM for Visualising Adult Material*

Though originally developed for imaging embryonic samples, protocols are already available for preparing biopsy material from adult individuals of various species for HREM imaging, as shown in Figure 3. Examples are materials stemming from rodents, zebrafish, turquoise killfish, ferrets and the fruit fly, for which there are preliminary data, but no publications yet exist.

**Figure 3.** HREM data created from material harvested from adult human (**A**–**C**) skin biopsies. Virtual resection perpendicular to the original HREM section plane (**A**). (**B**) shows details from the original HREM sections to demonstrate HREM's ability to visualize small dermal nerves (ne) and a Suquet-Hoyer canal (sh) in the top image and small dermal blood vessels (bv) in the bottom image. (**C**) shows a semitransparent 3D volume model of the sample combined with surface models of the lumina of the dermal arteries (red) and veins (blue). (**D**,**E**) show metastatic material (tu) in a liver (li) biopsy. Original 2D HREM section in (**D**). Boxed areas are zoomed-in views provided as inlays to the right. (**E**) shows a 3D volume model. ed, epidermis; de, dermis; hd, hypodermis; ca, fibrous tumour capsule; sd, sweat duct. Scale bars 500 μm in (**A**), (**C**), (**D**), (**E**); 150 μm in (**B**).

Published data does exist for the pig and the mouse, in which vascularisation in wound healing was researched [97,98], and for humans. In humans, HREM was successfully employed to develop a novel concept for dermal vascularisation in thick [99,100] and thin skin [101] and to analyse the topology of arteries and nerves in the auricle [102]. Besides this, it recently permitted the characterisation of plaques in the coronary arteries [103].

HREM could also show the structure of dermal matrix skin substitutes with and without seeded keratinocytes, and the effect of dermal matrix skin substitutes in combination with skin graft transplants on vascularisation in a porcine wound model. Recently, HREM was an integral part of a multimodal imaging pipeline for comprehensively characterising the vascularisation of murine tumour models [104].

#### *2.3. HREM for Other Organic Materials*

The materials primarily subjected to HREM imaging are sourced from animals. However, very recently, HREM expanded its application into the realm of plant sciences. More concretely, the morphology of wild-type and genetically altered tomato plants were visualised in order to study abnormal leaf axil patterning [105]. In pilot studies, the capacity of HREM for testing paper quality [28,106] and the fibre arrangement and the formation of keratinocytes seeded on skin replacement material [107] were evaluated. These experiments demonstrated that HREM is not restricted to biomedical research, but has yet-unknown capacities to aid research in many other fields of modern science.

#### **3. Conclusions and Further Perspectives**

HREM was developed in 2006, and for about a decade, operated on self-assembled apparatuses. In 2015, a fully operable, all-inclusive HREM machine became commercially available under the trade name of 3D Optical HREM imaging (OHREM). This increased the number of projects using the HREM technique, which was illustrated by a quadruplication of publications based on HREM data over the last ten years, and it boosted the development of new protocols and technical advancements. However, HREM is still to be considered as a technique in its beginnings with great potential for refinement and improvement. As pilot data has shown, it has a yet-unexplored high potential for visualising a broad variety of organic materials in unmatched resolution and data quality.

#### *3.1. Stitching*

One of the strengths of HREM is its ability to image volumes of up to 6 <sup>×</sup> <sup>6</sup> <sup>×</sup> 12 mm<sup>3</sup> in a numeric resolution of 3 <sup>×</sup> 3 <sup>×</sup> 3 <sup>μ</sup>m3. Increasing the numeric resolution is possible, but requires focusing on a smaller volume to be scanned [34,108]. First prototypes of HREM machines, which scan several images of the same block-surface and incorporate stitching algorithms for combining them, overcome these limitations and are already in use. It is to be expected that this promising approach will soon advance to become the HREM routine.

#### *3.2. Pipelines*

Even by using block scanning and stitching, the use of HREM is limited to exploring the microanatomy of specimens of a relatively small size. For large specimens, HREM imaging has to be combined with other imaging modalities to gain HREM detail in the context of overall specimen information. Since this is quite simple with techniques such μMRI, the first imaging pipeline that includes HREM dates back to the very early phase of HREM imaging [51]. Large batches of embryos were scanned with high-throughput μMRI in moderate resolution. Interesting specimens or specimen parts were then identified, selected and subjected to HREM imaging for providing tissue detail. This approach was modified and expanded in the last years [109], and the first publications providing protocols and demonstrating the benefits of high complex multimodal, multiscale imaging pipelines integrating HREM with imaging modalities such as μMRI, US, μCT, OCT, PAT and histopathology are already in preparation [104].

#### *3.3. Specific Stainings*

HREM data volumes comprise thousands of digital images, each resembling a greyscale image of a hematoxylin–eosine-stained histological section. Thus, HREM offers 3D information of morphological details of organic materials in a straightforward and simple way. Attempts to expand HREM to permit 3D visualisation of specifically labelled structures and molecular signals as well are as old as the HREM technique itself. Even the first publication included an example for visualising LacZ-stained tissues in mouse embryos and NBT/BCIP signals after whole-mount in situ hybridisation in zebrafish embryos [17]. However, despite these efforts, visualising specific stained tissues is still experimental and restricted to small specimens and specimens composed of loose and easy-to-penetrate tissues [103,110,111]. Protocols for late embryos and dense tissues are eagerly anticipated and will open new fields of applications for HREM. We are confident that with the growing community of HREM developers and users, solutions for this problem will be presented within the next few years.

**Author Contributions:** Conceptualization, S.H.G. and W.J.W.; writing—original draft preparation, S.H.G. and W.J.W.; writing—review and editing, S.H.G. and W.J.W.; visualization, S.H.G. and W.J.W.; supervision, W.J.W.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Barbara Maurer-Gesek and all the participants in the DMDD program. **Conflicts of Interest:** The authors declare no conflict of interest.
