Microvascular imaging of the unstained human superior colliculus using synchrotron-radiation phase-contrast microtomography

Abstract

Characterizing the microvasculature of the human brain is critical to advance understanding of brain vascular function. Most methods rely on tissue staining and microscopy in two-dimensions, which pose several challenges to visualize the three-dimensional structure of microvessels. In this study, we used an edge-based segmentation method to extract the 3D vasculature from synchrotron radiation phase-contrast microtomography (PC-μCT) of two unstained, paraffin-embedded midbrain region of the human brain stem. Vascular structures identified in PC-μCT were validated with histology of the same specimen. Using the Deriche-Canny edge detector that was sensitive to the boundary between tissue and vascular space, we could segment the vessels independent of signal variations in PC-μCT images. From the segmented volumetric vasculature, we calculated vessel diameter, vessel length and volume fraction of the vasculature in the superior colliculi. From high resolution images, we found the most frequent vessel diameter to be between 8.6–10.2 µm. Our findings are consistent with the known anatomy showing two types of vessels with distinctive morphology: peripheral collicular vessels and central collicular vessels. The proposed method opens up new possibilities for vascular research of the central nervous system using synchrotron radiation PC-μCT of unstained human tissue.

Protocol for 3D Virtual Histology of Unstained Human Brain Tissue using Synchrotron Radiation Phase-Contrast Microtomography

“…Quantitative MRI can be used to check the advancement of the fixation process. Moreover, MRI is useful for calculating the shrinkage ratio introduced by paraffin embedding, which is the next step (Wehrl et al, 2015, De Guzman et al, 2016, Lee et al et al, 2022, Nazemorroaya et al, 2022 The choice of the fixative agent will impact the tissue shrinkage ratio (Eckermann et al, 2021, Rodger et al, 2021 and tissue contrast in the resulting image (Strotton et al, 2018). For example, the fiber tract contrast is improved when tissue is fixed with 100 % ethanol compared to formalin (Rodger et al, 2021).…”

“…In SR PhC-µCT, these granules can be identified as hyperintense structures with a spherical shape (Fig 12,Fig S4) (Hieber et al, 2016). Recently, a 3D distribution of CA within the human brain stem was analysed from four subjects with mean age of 76 years old, reporting an increased density in the dorsomedial column of the periaqueductal gray (Lee et al, 2023). At the time being, the specificity of our CA detection method must be further investigated, since we anticipate that other densely packed spherical granules encountered in the brain such as Lewy bodies (Koh et al, 2006) may look similar to CA in SR PhC-µCT measurements.…”

“…(B) Segmented results from the region indicated with a black rectangle in panel A. Blood vessels (in red) were segmented using an edge-based approach (Lee et al, 2022). Corpora amylacea (yellow) were segmented using an intensity-based approach (Lee et al, 2023) and a similar approach was used to identify neuromelanin (cyan).…”

“…The brain stem acquired with 5 µm isotropic voxels (Fig. 8A) was processed using the edge-based vessel segmentation method detailed in Lee et al, (2022). The resulting vessel map (Fig.…”

“…Several studies have used SR PhC-µCT to investigate the microstructure of unstained human organs (Walsh et al, 2021, Einarsson et al, 2022, Lee et al, 2022). Some have acquired images from human brain samples with sub-cellular detail and successfully segmented neurons to study their three-dimensional spatial distributions (Hieber et al, 2006, Frost et al, 2023).…”