X-ray Micro-Computed Tomography for Nondestructive Three-Dimensional (3D) X-ray Histology.
ABSTRACT: Historically, micro-computed tomography (?CT) has been considered unsuitable for histologic analysis of unstained formalin-fixed, paraffin-embedded soft tissue biopsy specimens because of a lack of image contrast between the tissue and the paraffin. However, we recently demonstrated that ?CT can successfully resolve microstructural detail in routinely prepared tissue specimens. Herein, we illustrate how ?CT imaging of standard formalin-fixed, paraffin-embedded biopsy specimens can be seamlessly integrated into conventional histology workflows, enabling nondestructive three-dimensional (3D) X-ray histology, the use and benefits of which we showcase for the exemplar of human lung biopsy specimens. This technology advancement was achieved through manufacturing a first-of-kind ?CT scanner for X-ray histology and developing optimized imaging protocols, which do not require any additional sample preparation. 3D X-ray histology allows for nondestructive 3D imaging of tissue microstructure, resolving structural connectivity and heterogeneity of complex tissue networks, such as the vascular network or the respiratory tract. We also demonstrate that 3D X-ray histology can yield consistent and reproducible image quality, enabling quantitative assessment of a tissue's 3D microstructures, which is inaccessible to conventional two-dimensional histology. Being nondestructive, the technique does not interfere with histology workflows, permitting subsequent tissue characterization by means of conventional light microscopy-based histology, immunohistochemistry, and immunofluorescence. 3D X-ray histology can be readily applied to a plethora of archival materials, yielding unprecedented opportunities in diagnosis and research of disease.
Project description: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.
Project description:High-spatial-resolution histology of coronary artery autopsy samples play an important role for understanding heart disease such as myocardial infarction. Unfortunately, classical histology is often destructive, has thick slicing, requires extensive sample preparation, and is time-consuming. X-ray micro-CT provides fast nondestructive 3D imaging but absorption contrast is often insufficient, especially for observing soft-tissue features with high resolution. Here we show that propagation-based x-ray phase-contrast tomography has the resolution and contrast to image clinically relevant soft-tissue features in intact coronary artery autopsy samples with cellular resolution. We observe microscopic lipid-rich plaques, individual adipose cells, ensembles of few foam cells, and the thin fibrous cap. The method relies on a small-spot laboratory x-ray microfocus source, and provides high-spatial resolution in all three dimensions, fast data acquisition, minimum sample distortion and requires no sample preparation.
Project description:The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based ?CT (SR?CT) and classical ?CT. We demonstrate that SR?CT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SR?CT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases.
Project description:Advances in optical imaging modalities, such as optical coherence tomography (OCT), enable us to observe tissue microstructure at high resolution and in real time. Currently, core-needle biopsies are guided by external imaging modalities such as ultrasound imaging and x-ray computed tomography (CT) for breast and lung masses, respectively. These image-guided procedures are frequently limited by spatial resolution when using ultrasound imaging, or by temporal resolution (rapid real-time feedback capabilities) when using x-ray CT. One feasible approach is to perform OCT within small gauge needles to optically image tissue microstructure. However, to date, no system or core-needle device has been developed that incorporates both three-dimensional OCT imaging and tissue biopsy within the same needle for true OCT-guided core-needle biopsy. We have developed and demonstrate an integrated core-needle biopsy system that utilizes catheter-based 3-D OCT for real-time image-guidance for target tissue localization, imaging of tissue immediately prior to physical biopsy, and subsequent OCT imaging of the biopsied specimen for immediate assessment at the point-of-care. OCT images of biopsied ex vivo tumor specimens acquired during core-needle placement are correlated with corresponding histology, and computational visualization of arbitrary planes within the 3-D OCT volumes enables feedback on specimen tissue type and biopsy quality. These results demonstrate the potential for using real-time 3-D OCT for needle biopsy guidance by imaging within the needle and tissue during biopsy procedures.
Project description:Background:Along with the conventional deposition of physical types at natural history museums, the deposition of 3-dimensional (3D) image data has been proposed for rare and valuable museum specimens, such as irreplaceable type material. Findings:Micro computed tomography (?CT) scan data of 5 hermit crab species from South Africa, including rare specimens and type material, depicted main identification characteristics of calcified body parts. However, low-image contrasts, especially in larger (>50 mm total length) specimens, did not allow sufficient 3D reconstructions of weakly calcified and fine characteristics, such as soft tissue of the pleon, mouthparts, gills, and setation. Reconstructions of soft tissue were sometimes possible, depending on individual sample and scanning characteristics. The raw data of seven scans are publicly available for download from the GigaDB repository. Conclusions:Calcified body parts visualized from ?CT data can aid taxonomic validation and provide additional, virtual deposition of rare specimens. The use of a nondestructive, nonstaining ?CT approach for taxonomy, reconstructions of soft tissue structures, microscopic spines, and setae depend on species characteristics. Constrained to these limitations, the presented dataset can be used for future morphological studies. However, our virtual specimens will be most valuable to taxonomists who can download a digital avatar for 3D examination. Simultaneously, in the event of physical damage to or loss of the original physical specimen, this dataset serves as a vital insurance policy.
Project description:<h4>Background and aims</h4>Visualization of arterial lesions in situ can enhance understanding of atherosclerosis progression and potentially improve experimental therapies. Conventional histology methods for assessing atherosclerotic lesions are robust but are destructive and may prevent further tissue analysis. The objective of the current study was to evaluate a novel, nondestructive method for visualization and characterization of atherosclerotic lesions as an alternative or complementary to routine histology. Thus, we tested the hypothesis that micro-computed tomography (micro-CT) paired with an iodine-based radiopaque stain would effectively characterize atherosclerotic plaques in a manner comparable to routine histology while maintaining sample integrity and providing whole-volume data.<h4>Methods</h4>We examined porcine coronary arteries with varying degrees of atherosclerosis, using micro-CT in the absence and presence of iohexol (240 mgI/ml). Following iohexol washout, routine histological assessment of the samples was performed with hematoxylin and eosin and Masson's trichrome.<h4>Results</h4>Iohexol staining generated soft tissue delineation and subsequent atherosclerotic plaque assessment via augmented radiopacity, permitting three-dimensional (3D) reconstruction of these lesions, maintaining in situ architecture. Although plaque distribution and arterial wall tissue layers were discernible, micro-CT was incapable of discriminating cell types comprising the plaque. Calcium phosphate deposition was readily located and visualized in 3D space, independent of iohexol.<h4>Conclusions</h4>The results of this study establish micro-CT, combined with a diffusible radiopaque contrast agent, as a powerful imaging modality for visualizing in situ architecture of atherosclerotic plaques. Our findings demonstrate that micro-CT can be used to identify plaque distribution and calcium deposition complementary to routine histological analysis.
Project description:Imaging mass spectrometry is becoming a key technology for the investigation of the molecular content of biological tissue sections in direct correlation with the underlying histology. Much of our work has been done with fresh-frozen tissue sections that has undergone minimal protein degradation between the time a tissue biopsy is sampled and the time it is snap-frozen so that no preserving or fixing agents need to be added to the frozen biopsy. However, in many sampling environments, immediate flash freezing may not be possible and so we have explored the use of ethanol-preserved, paraffin-embedded tissue specimens for proteomic analyses. Solvent-only preserved tissue specimens provide long-term preservation at room temperature, generation of high quality histological sections and little if any chemical alteration of the proteins. Using mouse organs, several key steps involved in the tissue dehydration process have been investigated to assess the potential of such preserved specimens for profiling and imaging mass spectrometry investigations.
Project description:Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.
Project description:Prostate cancer treatment planning is largely dependent upon examination of core-needle biopsies. The microscopic architecture of the prostate glands forms the basis for prognostic grading by pathologists. Interpretation of these convoluted three-dimensional (3D) glandular structures via visual inspection of a limited number of two-dimensional (2D) histology sections is often unreliable, which contributes to the under- and overtreatment of patients. To improve risk assessment and treatment decisions, we have developed a workflow for nondestructive 3D pathology and computational analysis of whole prostate biopsies labeled with a rapid and inexpensive fluorescent analogue of standard hematoxylin and eosin (H&E) staining. This analysis is based on interpretable glandular features and is facilitated by the development of image translation-assisted segmentation in 3D (ITAS3D). ITAS3D is a generalizable deep learning-based strategy that enables tissue microstructures to be volumetrically segmented in an annotation-free and objective (biomarker-based) manner without requiring immunolabeling. As a preliminary demonstration of the translational value of a computational 3D versus a computational 2D pathology approach, we imaged 300 <i>ex vivo</i> biopsies extracted from 50 archived radical prostatectomy specimens, of which, 118 biopsies contained cancer. The 3D glandular features in cancer biopsies were superior to corresponding 2D features for risk stratification of patients with low- to intermediate-risk prostate cancer based on their clinical biochemical recurrence outcomes. The results of this study support the use of computational 3D pathology for guiding the clinical management of prostate cancer. SIGNIFICANCE: An end-to-end pipeline for deep learning-assisted computational 3D histology analysis of whole prostate biopsies shows that nondestructive 3D pathology has the potential to enable superior prognostic stratification of patients with prostate cancer.
Project description:OBJECTIVE:To develop an approach for radiology-pathology fusion of ex vivo histology of surgically excised pulmonary nodules with pre-operative CT, to radiologically map spatial extent of the invasive adenocarcinomatous component of the nodule. METHODS:Six subjects (age: 75?±?11 years) with pre-operative CT and surgically excised ground-glass nodules (size: 22.5?±?5.1 mm) with a significant invasive adenocarcinomatous component (>5 mm) were included. The pathologist outlined disease extent on digitized histology specimens; two radiologists and a pulmonary critical care physician delineated the entire nodule on CT (in-plane resolution: <0.8 mm, inter-slice distance: 1-5 mm). We introduced a novel reconstruction approach to localize histology slices in 3D relative to each other while using CT scan as spatial constraint. This enabled the spatial mapping of the extent of tumour invasion from histology onto CT. RESULTS:Good overlap of the 3D reconstructed histology and the nodule outlined on CT was observed (65.9?±?5.2%). Reduction in 3D misalignment of corresponding anatomical landmarks on histology and CT was observed (1.97?±?0.42 mm). Moreover, the CT attenuation (HU) distributions were different when comparing invasive and in situ regions. CONCLUSION:This proof-of-concept study suggests that our fusion method can enable the spatial mapping of the invasive adenocarcinomatous component from 2D histology slices onto in vivo CT. KEY POINTS:• 3D reconstructions are generated from 2D histology specimens of ground glass nodules. • The reconstruction methodology used pre-operative in vivo CT as 3D spatial constraint. • The methodology maps adenocarcinoma extent from digitized histology onto in vivo CT. • The methodology potentially facilitates the discovery of CT signature of invasive adenocarcinoma.