Ethanol fixation method for heart and lung imaging in micro-CT

Patzelt, Matej; Mrzílková, Jana; Dudák, Jan; Krejci, F.; Žemlička, J.; Karch, Jakub; Musil, Vladimír; Rosina, Jozef; Sýkora, V.; Horehledova, Barbora; Zach, Petr

doi:10.1007/s11604-019-00830-6

Cited by 18 publications

(27 citation statements)

References 27 publications

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“…cro-CT setups, contrast is produced by the absorption of X-rays traversing the tissue. Lung tissue (soft tissue) exhibits relatively low and homogenous absorption levels and therefore yields low contrast volumetric data, unless stained with contrast-enhancing agents before image acquisition (8,31). The low contrast limits the ability to resolve fine detail and makes the acquired image data less comparable to histology and immunohistochemistry.…”

Section: Introductionmentioning

confidence: 99%

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia

Norvik

Westöö

Peruzzi

et al. 2020

American Journal of Physiology-Lung Cellular and Molecular Phys

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This study aimed to explore the value of synchrotron-based phase-contrast microcomputed tomography (micro-CT) in pulmonary vascular pathobiology. The microanatomy of the lung is complex with intricate branching patterns. Tissue sections are therefore difficult to interpret. Recruited intrapulmonary bronchopulmonary anastomoses (IBAs) have been described in several forms of pulmonary hypertension, including alveolar capillary dysplasia with misaligned pulmonary veins (ACD/MPV). Here, we examine paraffin-embedded tissue using this nondestructive method for high-resolution three-dimensional imaging. Blocks of healthy and ACD/MPV lung tissue were used. Pulmonary and bronchial arteries in the ACD/MPV block had been preinjected with dye. One section per block was stained, and areas of interest were marked to allow precise beam-alignment during image acquisition at the X02DA TOMCAT beamline (Swiss Light Source). A ×4 magnifying objective coupled to a 20-µm thick scintillating material and a sCMOS detector yielded the best trade-off between spatial resolution and field-of-view. A phase retrieval algorithm was applied and virtual tomographic slices and video clips of the imaged volumes were produced. Dye injections generated a distinct attenuation difference between vessels and surrounding tissue, facilitating segmentation and three-dimensional rendering. Histology and immunohistochemistry post-imaging offered complementary information. IBAs were confirmed in ACD/MPV, and the MPVs were positioned like bronchial veins/venules. We demonstrate the advantages of using synchrotron-based phase-contrast micro-CT for three-dimensional characterization of pulmonary microvascular anatomy in paraffin-embedded tissue. Vascular dye injections add additional value. We confirm intrapulmonary shunting in ACD/MPV and provide support for the hypothesis that MPVs are dilated bronchial veins/venules.

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Section: Introductionmentioning

confidence: 99%

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia

Norvik

Westöö

Peruzzi

et al. 2020

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…We have already scanned hearts in PBS/Formalin. The challenge here is the much weaker contrast compared to dehydrated samples, see also [43], which requires higher propagation distance and careful optimization of photon energy.…”

Section: Discussionmentioning

confidence: 99%

Multi-scale X-ray phase-contrast tomography of murine heart tissue

Reichardt¹,

Frohn²,

Khan³

et al. 2020

Biomed. Opt. Express

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The spatial organization of cardiac muscle tissue exhibits a complex structure on multiple length scales, from the sarcomeric unit to the whole organ. Here we demonstrate a multi-scale three-dimensional imaging (3d) approach with three levels of magnification, based on synchrotron X-ray phase contrast tomography. Whole mouse hearts are scanned in an undulator beam, which is first focused and then broadened by divergence. Regions-of-interest of the hearts are scanned in parallel beam as well as a biopsy by magnified cone beam geometry using a X-ray waveguide optic. Data is analyzed in terms of orientation, anisotropy and the sarcomeric periodicity via a local Fourier transformation.

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“…The noise could be suppressed by a longer acquisition time and the contrast enhanced by a longer incubation in ethanol. 20 To keep the results comparable, experiments with higher acquisition times or incubation in ethanol are not shown in this study. For the preparation of the samples in liquid, it is mandatory to prevent bubbles because even small bubbles in the liquid can grow and cause motion artifacts.…”

Section: Evaluation Of the Tomographic Reconstructionsmentioning

confidence: 99%

“…1. paraffin embedding, 18 2. paraffin embedding with an iodine stain, 6 3. paraffin embedding with a phosphotungstic acid (PTA) stain, 18,19 4. embedding in liquid ethanol, 20 and 5. evaporation-of-solvent (EOS) method. 21 The whole explanted hearts were washed in phosphate-buffered saline and fixated in 4% formaldehyde solution for 24 h. The stains were administered in solution by diffusion of the staining agent, with incubation times of 24 h (iodine) or 7 days (PTA).…”

mentioning

confidence: 99%

Fiber orientation in a whole mouse heart reconstructed by laboratory phase-contrast micro-CT

et al. 2020

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Purpose: We present a phase-contrast x-ray tomography study of wild type C57BL/6 mouse hearts as a nondestructive approach to the microanatomy on the scale of the entire excised organ. Based on the partial coherence at a home-built phase-contrast μ-CT setup installed at a liquid metal jet source, we exploit phase retrieval and hence achieve superior image quality for heart tissue, almost comparable to previous synchrotron data on the whole organ scale. Approach: In our work, different embedding methods and heavy metal-based stains have been explored. From the tomographic reconstructions, quantitative structural parameters describing the three-dimensional (3-D) architecture have been derived by two different fiber tracking algorithms. The first algorithm is based on the local gradient of the reconstructed electron density. By performing a principal component analysis on the local structure-tensor of small subvolumes, the dominant direction inside the volume can be determined. In addition to this approach, which is already well established for heart tissue, we have implemented and tested an algorithm that is based on a local 3-D Fourier transform. Results: We showed that the choice of sample preparation influences the 3-D structure of the tissue, not only in terms of contrast but also with respect to the structural preservation. A heart prepared with the evaporation-of-solvent method was used to compare both algorithms. The results of structural orientation were very similar for both approaches. In addition to the determination of the fiber orientation, the degree of filament alignment and local thickness of single muscle fiber bundles were obtained using the Fourier-based approach. Conclusions: Phase-contrast x-ray tomography allows for investigating the structure of heart tissue with an isotropic resolution below 10 μm. The fact that this is possible with compact laboratory instrumentation opens up new opportunities for screening samples and optimizing sample preparation, also prior to synchrotron beamtimes. Further, results from the structural analysis can help in understanding cardiovascular diseases or can be used to improve computational models of the heart.

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Ethanol fixation method for heart and lung imaging in micro-CT

Cited by 18 publications

References 27 publications

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia

Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia

Multi-scale X-ray phase-contrast tomography of murine heart tissue

Fiber orientation in a whole mouse heart reconstructed by laboratory phase-contrast micro-CT

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