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A simple nuclear contrast staining method for microCT-based 3D histology using lead(II) acetate.

ABSTRACT: X-ray microtomography (microCT) enables histological-scale 3D imaging of many types of biological samples, but it has yet to rival traditional histology for differentiation of tissue types and cell components. This report presents prima facie results indicating that a simple lead(II) acetate staining solution can impart preferential X-ray contrast to cell nuclei. While not strictly selective for nuclei, the staining reflects local cell-density differences. It can be applied in a single overnight treatment and does not require hematoxylin staining or drying of the sample. The stain is removable with EDTA, and it may enhance early calcifications. A basic protocol is given as a guide for further testing and optimization.

SUBMITTER: Metscher B

PROVIDER: S-EPMC7930760 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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A simple nuclear contrast staining method for microCT-based 3D histology using lead(II) acetate.

Metscher Brian B

Journal of anatomy 20201102 4

X-ray microtomography (microCT) enables histological-scale 3D imaging of many types of biological samples, but it has yet to rival traditional histology for differentiation of tissue types and cell components. This report presents prima facie results indicating that a simple lead(II) acetate staining solution can impart preferential X-ray contrast to cell nuclei. While not strictly selective for nuclei, the staining reflects local cell-density differences. It can be applied in a single overnight ...[more]

PMID: 33140846

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Project description:PurposeThe purpose of this paper is to validate a rapid and cost-effective ex vivo technique, microCT-based virtual histology, as an alternative to MRI imaging for assessing the therapeutic response in genetically engineered mouse models of cancer.ProceduresAll animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at San Antonio. MRI imaging was performed on 6-week-old, bortezomib-treated genetically engineered Patched1, p53 mice that recapitulate the characteristics of human medulloblastoma. After MRI scans, the same mice were euthanized to collect brain or spine samples for virtual histology staining followed by microCT scanning.ResultsNine-micrometer resolution ex vivo micro X-ray computed tomography (microCT)-based virtual histology images were qualitatively reflective of high-field live animal images obtained with magnetic resonance imaging (MRI) and histopathology. Cerebellar volumes on microCT-based virtual histology correlated closely with MRI cerebellar volumes (R = 0.998). MRI and microCT-based virtual histology both indicated a significant difference between cerebellar volumes of untreated and treated mice (p = 0.02 and p = 0.04, respectively). The ex vivo microCT method also allowed a 7,430-fold improvement in voxel resolution (voxel volume of 729 μm³ for 9-μm isometric resolution microCT vs. 5,416,800 μm³ for 400 × 111 × 122 μm resolution MRI) at a 28% cost savings ($400 vs. $555 per animal).ConclusionThe ex vivo, en bloc technique of microCT-based virtual histology matched MRI in reflecting histopathology. MicroCT-based virtual histology proved to be a more cost-effective technique and less labor-intensive. On the other hand, MRI provides ability to perform in vivo imaging, faster scanning and lower radiation dose by sacrificing the spatial resolution. Thus, both in vivo MRI and ex vivo microCT-based virtual histology are effective means of quantitatively evaluating therapeutic response in preclinical models of cerebellar tumors including the childhood cancer, medulloblastoma.

Project description:Current breast tumor margin detection methods are destructive, time-consuming, and result in significant reoperative rates. Dual-modality photoacoustic tomography (PAT) and ultrasound has the potential to enhance breast margin characterization by providing clinically relevant compositional information with high sensitivity and tissue penetration. However, quantitative methods that rigorously compare volumetric PAT and ultrasound images with gold-standard histology are lacking, thus limiting clinical validation and translation. Here, we present a quantitative multimodality workflow that uses inverted Selective Plane Illumination Microscopy (iSPIM) to facilitate image co-registration between volumetric PAT-ultrasound datasets with histology in human invasive ductal carcinoma breast tissue samples. Our ultrasound-PAT system consisted of a tunable Nd:YAG laser coupled with a 40 MHz central frequency ultrasound transducer. A linear stepper motor was used to acquire volumetric PAT and ultrasound breast biopsy datasets using 1100 nm light to identify hemoglobin-rich regions and 1210 nm light to identify lipid-rich regions. Our iSPIM system used 488 nm and 647 nm laser excitation combined with Eosin and DRAQ5, a cell-permeant nucleic acid binding dye, to produce high-resolution volumetric datasets comparable to histology. Image thresholding was applied to PAT and iSPIM images to extract, quantify, and topologically visualize breast biopsy lipid, stroma, hemoglobin, and nuclei distribution. Our lipid-weighted PAT and iSPIM images suggest that low lipid regions strongly correlate with malignant breast tissue. Hemoglobin-weighted PAT images, however, correlated poorly with cancerous regions determined by histology and interpreted by a board-certified pathologist. Nuclei-weighted iSPIM images revealed similar cellular content in cancerous and non-cancerous tissues, suggesting malignant cell migration from the breast ducts to the surrounding tissues. We demonstrate the utility of our nondestructive, volumetric, region-based quantitative method for comprehensive validation of 3D tomographic imaging methods suitable for bedside tumor margin detection.

Dataset Information

A simple nuclear contrast staining method for microCT-based 3D histology using lead(II) acetate.

Publications

A simple nuclear contrast staining method for microCT-based 3D histology using lead(II) acetate.

Similar Datasets

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