Nucleus-specific X-ray stain for 3D virtual histology

Abstract

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Scalable method for micro-CT analysis enables large scale quantitative characterization of brain lesions and implants

“…We wanted to avoid dehydration of the tissue and embedding in resin or paraffin as these techniques often lead to substantial shrinkage of tissue 29 , 30 . We therefore adapted protocols using eosin 27 , 28 to provide enhanced contrast within the brain, and imaged the brain using monochromatic X-rays at the Advanced Light Source (ALS) at Lawrence Berkeley National Lab.…”

“…We adapt decalcification techniques 25 , 26 to image the brain with critical parts of the skull intact. Furthermore, we avoid any dehydration or embedding of the tissue, and enhance contrast to the post-mortem brain using eosin as an additional contrast agent 27 , 28 . The resulting protocol allows for evaluation of brain structure relative to the coordinates determined by skull sutures and permits further processing with standard histologic techniques.…”

A tube-source X-ray microtomography approach for quantitative 3D microscopy of optically challenging cell-cultured samples

“…These kinds of samples include fibrous and porous tissue-engineering scaffolds and organ-on-a-chip constructs with open channels and cavities. To modify the µCT-based methodological pipeline for studying more confluent 3D samples, semi-specific labeling based on more diffusive agents could be used to substitute the antibodies, such as using hematein lead(II) complex for labeling nuclei in tissues 11 . A short introduction to available contrasting methods for various samples and target structures is available in Supplementary Note 2.…”

A Simple Nuclear Contrast Staining Method for MicroCT-Based 3D Histology Using Lead(II) Acetate

“…The staining solution was lead(II) acetate trihydrate (Pb(CH 3 COO) 2 •3H 2 O, CAS 6080-56-4; Sigma-Aldrich 467863) dissolved in distilled water at 20% w/v (0.53M, pH~5.5). This solution was stored at room temperature and could be used for at least two weeks (up to four weeks according to Müller et al (2018)). Concentrations of 10%, 2%, and 1% were also prepared in distilled water.…”