Abstract
Advancements in tissue visualization techniques have spurred significant gains in the biomedical sciences by enabling researchers to integrate their datasets across anatomical scales. Of particular import are techniques that enable the interpolation of multiple hierarchical scales in samples taken from the same individuals. In this study, we demonstrate that two-dimensional histology techniques can be employed on neural tissues following three-dimensional diffusible iodine-based contrast-enhanced computed tomography (diceCT) without causing tissue degradation. This represents the first step toward a multiscale pipeline for brain visualization. We studied brains from adolescent male Sprague-Dawley rats, comparing experimental (diceCT-stained then de-stained) to control (without diceCT) brains to examine neural tissues for immunolabeling integrity, compare somata sizes, and distinguish neurons from glial cells within the telencephalon and diencephalon. We hypothesized that if experimental and control samples do not differ significantly in morphological cell analysis, then brain tissues are robust to the chemical, temperature, and radiation environments required for these multiple, successive imaging protocols. Visualizations for experimental brains were first captured via micro-computed tomography scanning of isolated, iodine-infused specimens. Samples were then cleared of iodine, serially sectioned, and prepared again using immunofluorescent, fluorescent, and cresyl violet labeling, followed by imaging with confocal and light microscopy, respectively. Our results show that many neural targets are resilient to diceCT imaging and compatible with downstream histological staining as part of a low-cost, multiscale brain imaging pipeline.
Highlights
Scientific gains in understanding biological organization and complexity are closely associated with technological and methodological gains in bio-imaging
This study demonstrates that two-dimensional histology techniques can be employed on neural tissues following three-dimensional diffusible iodine-based contrast-enhanced computed tomography without causing tissue degradation
We propose the use of diffusible iodine-based contrast-enhanced computed tomography (diceCT) (Gignac et al 2016), which is commonly employed in organismal biology research, as a novel, inexpensive, and high-resolution ex vivo alternative to μMRI
Summary
Scientific gains in understanding biological organization and complexity are closely associated with technological and methodological gains in bio-imaging. Visualization tools widely used in biomedical and clinical imaging, such as light-sheet microscopy (Voie et al 1993), magnetic resonance imaging (MRI: Jackson and Langham 1968; Lauterbur 1974; Damadian et al 1976; Henshaw et al 1977; μMRI: Smith et al 1996; Dodd et al 1999), white matter tractography (diffusion tensor image analysis; Basser et al 1994; Jellison et al 2004), X-ray micro-computed tomography (μCT; Hounsfield 1977; Feldkamp et al 1989; Rüegsegger et al 1996), and positron emission tomography (Ter-Pogossian et al 1975; Phelps et al 1976; Muehllehner et al 1976; Hawkins et al 1992; Jones and Townsend 2017), are continuously refined to allow investigators to improve the visualization of gross anatomical, microscopic, and cellularlevel structure and function of biological tissues These methodological enhancements have spurred significant advancement within the biomedical sciences by enabling researchers to integrate datasets across anatomical scales––from the low-resolution capture of gross structures to the fine-resolution capture of subcellular cytoskeletal elements. Iodine absorbs X-rays and binds preferentially to sugars and fatty tissues, making it excellent for nervous system visualization due to its clear differentiation of myelinated and non-myelinated structures at micron-scale resolution using μCT (Gignac and Kley 2018)
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