Scatter labeled imaging of microvasculature in excised tissue (SLIME)
Inventors
Jenkins, Michael W. • LIU, Yehe
Assignees
Case Western Reserve University • National Institutes of Health NIH • University Hospitals Cleveland Medical Center • US Department of Health and Human Services
Publication Number
US-11845955-B2
Publication Date
2023-12-19
Expiration Date
2038-11-09
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Abstract
The present disclosure relates to a simple, fast, and low cost method for 3D microvascular imaging, termed “scatter labeled imaging of microvasculature in excised tissue” (SLIME). The method can include perfusing a contrast agent through vasculature of a tissue sample. The contrast agent can include colloids and a dispersant. After the contrast agent is perfused through the vasculature, the vasculature of the tissue sample can be treated with a molecule that cross links with at least a portion of the dispersant to form a sticky, non-Newtonian polymer that prevents leakage of the contrast agent out of the vasculature of the tissue sample. The tissue sample can then be immersed in a solution comprising a clearing agent and subsequently imaged.
Core Innovation
The present disclosure provides a simple, fast, and low cost method for three-dimensional microvascular imaging of excised tissue, termed "scatter labeled imaging of microvasculature in excised tissue" (SLIME). SLIME combines the use of an optical coherence tomography (OCT) contrast agent and optical clearing, enabling high speed, high resolution, and wide field of view imaging of microvasculature. The method includes perfusing a tissue sample's vasculature with a contrast agent composed of colloids and a dispersant, followed by treating the tissue with a molecule that cross-links with the dispersant to form a sticky, non-Newtonian polymer that prevents leakage of the contrast agent from the vasculature.
After this cross-linking, the tissue sample is immersed in a solution containing a clearing agent modified to contain boric acid, which optically clears the tissue by reducing light scattering and increasing imaging depth, without affecting the contrast agent. This combination produces high contrast between the vascular component and surrounding tissue, allowing volumetric image data acquisition and subsequent processing and analysis for visualization and quantification of microvascular morphology. SLIME is applicable to various excised tissues and biological lumens which can be perfused, extending the vascular imaging capabilities of OCT in a cost effective and time-efficient manner.
The problem being addressed is that existing three-dimensional microvascular mapping methods, such as micro-CT and confocal microscopy, are often complex, expensive, time consuming, and ill-suited for large volume samples and large population studies, such as phenotyping and drug screening. SLIME provides an improved methodology that overcomes these limitations by enabling rapid, cost effective, high resolution, and wide field of view 3D microvascular imaging with minimal sample preparation complexity.
Claims Coverage
The patent includes one independent claim presenting a method with multiple inventive features focused on a contrast agent perfusion, cross-linking to prevent leakage, optical clearing, imaging, and image processing steps.
Colloidal-based contrast agent perfusion through tissue component
The method involves perfusing a colloidal-based contrast agent that includes a dispersant through a component of a tissue sample to fill the component with the contrast agent, maintaining a low viscosity suitable for perfusion.
Cross-linking agent treatment to prevent leakage
The tissue sample is treated with a cross-linking agent that chemically cross-links with the dispersant to form a sticky, non-Newtonian polymer which clogs openings in the tissue component and prevents leakage of the contrast agent.
Performing optical clearing with a modified clearing agent
Optical clearing is performed on the tissue sample by immersion in a solution comprising a clearing agent modified to contain boric acid, which enhances tissue transparency and further stabilizes the contrast agent inside the tissue.
Imaging the prepared tissue sample to generate high contrast images
One or more images of the tissue sample are recorded, particularly using optical coherence tomography (OCT) imaging, where the colloidal contrast agent (e.g., TiO2 nanoparticles) provides strong signal and contrast with the surrounding tissue.
Image processing to enhance quality and perform quantitative analysis
The image processing includes applying multi-iterative 3D median filtering for noise reduction, coherence-enhanced diffusion filtering for noise reduction and vessel continuity improvement, background subtraction and 3D unsharp filtering for contrast enhancement, and segmentation with skeletonization into 3D graph data for quantitative analysis.
Assessment of disease state and morphology quantification
Analyzing the processed images allows assessing whether a disease state exists and quantifying morphological aspects of the tissue component, such as vessel length density and local tortuosity.
The claims fully cover the novel method of perfusing a colloidal-based contrast agent in tissue, applying cross-linking to prevent leakage, optical clearing with a boric acid-containing agent, imaging (especially with OCT), and detailed image processing to analyze the microvascular morphology and disease states.
Stated Advantages
SLIME is a simple, fast, and low cost method for 3D microvascular imaging compared to existing methods.
The method allows high resolution, wide field of view imaging with high speed, enabling large volume and large cohort studies.
SLIME overcomes common imaging artifacts such as shadowing and provides greater imaging depth limited only by the optics.
The optical clearing step combined with a stable contrast agent enhances imaging depth and contrast without affecting the contrast agent.
The image processing pipeline provides effective noise reduction, vessel continuity, and quantitative morphological analysis capabilities.
Documented Applications
3D microvascular imaging of excised tissues for evaluation of microvascular morphology across large fields of view.
Use in large cohort studies requiring assessment of vascular morphology and organization in animal models.
Imaging vascular abnormalities in disease models, for example, fetal alcohol syndrome in quail embryo hearts.
Quantitative measurements of microvascular features such as vessel length density, vessel tortuosity, and vessel length to specific points using skeletonized 3D images.
Evaluation of blood vessels and other perfusable biological lumens including lymphatic and digestive tracts.
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