###### Abstract  
High-resolution episodic microscopy (HREM) is an imaging technique that permits the simple and rapid generation of three-dimensional (3D) digital volume data of histologically embedded and physically sectioned specimens. The data can be immediately used for high-detail 3D analysis of a broad variety of organic materials with all modern methods of 3D visualisation and display. Since its first description in 2006, HREM has been adopted as a method for exploring organic specimens in many fields of science, and it has recruited a slowly but steadily growing user community. This review aims to briefly introduce the basic principles of HREM data generation and to provide an overview of scientific publications that have been published in the last 13 years involving HREM imaging. The studies to which we refer describe technical details and specimen-specific protocols, and provide examples of the successful use of HREM in biological, biomedical and medical research. Finally, the limitations, potentials and anticipated further improvements are briefly outlined.  
imaging; 3D; high-resolution episodic microscopy; episodic; phenotyping; HREM +
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HREM [17] is an episodic imaging method which generates a series of inherently aligned digital images of histologically processed and embedded specimens. The images are virtually stacked to be analysed by scrolling through the original or virtual resections and to lend themselves to volume rendering or segmentation and surface rendering. Thus, HREM is in line with other episodic imaging techniques, such as serial block-face scanning electron microscopy (SBFSEM) [18], serial reconstruction technique [19; 20; 21], Epi-3D [22], surface imaging microscopy [23; 24], imaging cryomicrotome [25], serial block-face imaging [26] and episodic fluorescent image capturing (EFIC) [27], although it is optimised for a low microscopic level of resolution.  
In a nutshell, materials are harvested and processed as for traditional histology. During dehydration, they are stained with eosin red mixtures, and resin (JB4) dyed with eosin or eosin/acridine orange is used for infiltration and as an embedding material. The resulting resin blocks are sectioned on a microtome, while digital images of subsequently exposed block surfaces are captured in fluorescence-mode using yellow fluorescent protein (YFP) (excitation 500/20 nm, emission 535/30 nm) or green fluorescent protein (GFP) (excitation 470/40, emission 525/50) filter sets for visualising eosin-contrasted structures. Cells and tissues specifically stained with LacZ or NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolynphosphate) appear heavily contrasted when also using a Texas Red filter system as shown in Figure 1. Several protocol variations optimised for various materials are already published and extensively discussed [28; 29; 30; 31; 32].  
Figure 1: Workflow. The flow chart briefly summarizes the steps of the HREM-workflow from sample harvesting to 3D visualisation and data analysis.  
HREM data typically have a voxel size of \(2\times 2\times 2\)\(\upmu\)m\({}^{3}\) and are created from specimens with a volume of \(6\times 6\times 6\) mm\({}^{3}\), although larger and smaller samples and higher or lower resolutions are possible. Data contrasts fit for identifying cells and nuclei, nerve fibres, capillaries and collagen bundles in the context of overall morphology and tissue architecture. Data generation varies according to specimen size, but it takes approximately 4-5 h for data comprised of 2000 single section images. Since numerous publications comprehensively describe the method [33; 34; 35; 36] and ready-to-go HREM apparatuses are already commercially available (OHREM, Indigo Scientific Ltd., Baldock, England), this paper will not provide protocols or technical descriptions, but will focus on discussing published scientific results and potential applications.