Systems and methods for imaging and processing tissue
Inventors
Assignees
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Publication Number
US-10908087-B2
Publication Date
2021-02-02
Expiration Date
Abstract
In accordance with preferred embodiments of the present invention, a method for imaging tissue, for example, includes the steps of mounting the tissue on a computer controlled stage of a microscope, determining volumetric imaging parameters, directing at least two photons into a region of interest, scanning the region of interest across a portion of the tissue, imaging a plurality of layers of the tissue in a plurality of volumes of the tissue in the region of interest, sectioning the portion of the tissue, capturing the sectioned tissue, and imaging a second plurality of layers of the tissue in a second plurality of volumes of the tissue in the region of interest, and capturing each sectioned tissue, detecting a fluorescence image of the tissue due to said excitation light; and processing three-dimensional data that is collected to create a three-dimensional image of the region of interest. Further, captured tissue sections can be processed, re-imaged, and indexed to their original location in the three dimensional image.
Core Innovation
The invention provides automated imaging and storage of tissue samples by illuminating a first region of tissue with a multiphoton imaging microscope, detecting light to form an image, and sectioning a first portion of the imaged tissue in a fluid bath to expose a second region of tissue. The method transports the sectioned first portion with a section capture and transport device positioned at the sectioning device to remove sectioned portions from the fluid bath so that individual sections are delivered for storage. The delivered sections are serially indexed and stored for further processing, and the workflow repeats for a second region of tissue.
After imaging and sectioning for each tissue region, the method further processes at least one transported section to generate processed tissue section data and analyzes the processed tissue section data in combination with an image of at least one region of tissue. In the automated tissue imaging, sectioning and transport variants, a data processor records an indexed position for each individual transported section to enable further processing, and the further processing generates tissue section data for each further processed indexed tissue section. The method includes controlling a sectioning device to section imaged tissue portions in a fluid bath and transporting the sectioned portions with a transport system to provide serially indexed transported sections.
The documented approach includes generating second images of at least one region of tissue during further processing, where the image of the at least one region comprises an optical tomography image, a multiphoton image, a widefield image, a confocal image, or a time resolved fluorescence image. The description further emphasizes capturing and storing individual sections via serial indexing, enabling further processing and re-imaging, and indexing/registration back to the original 3D whole-mount dataset using overlap-based digital registration/morphing. The stated motivation relates to TPM depth/FOV/speed limits and to the difficulty of whole-mount immunohistochemistry, and the system aims to reduce sectioning artifacts via pre-imaged cut planes and provide accurate registration using overlapping volumes.
Claims Coverage
The independent claims cover automated workflows for multiphoton imaging of first and second tissue regions, serial sectioning in a fluid bath, transport to serially indexed storage, and subsequent further processing with data generation and analysis using indexed images and/or indexed position recording. The coverage spans two independent methods and identifies the core inventive features as imaging-first-region, fluid-bath sectioning, serial indexing storage via a section capture and transport device, and further processing with imaging modalities and/or analysis using region images.
Automated imaging, serial indexing, and storage with multiphoton pre-images and further processing analysis
Illuminating a first region of tissue with a multiphoton imaging microscope to form an image, sectioning a first portion in a fluid bath to expose a second region and forming a sectioned first portion, transporting the sectioned first portion to deliver a first transported section for storage with individual sections serially indexed and stored for further processing, illuminating the second region and detecting light to form an image, sectioning a second portion to form a sectioned second portion, transporting the second portion to deliver a second transported section for storage with the first and second transported sections serially indexed, further processing at least one transported section to generate processed tissue section data, and analyzing the processed tissue section data in combination with an image of at least one region of tissue.
Automated tissue imaging, sectioning and transport with processor-recorded indexed positions and further processed image data
Illuminating a first region of tissue and detecting light to form an image of the first region, controlling a sectioning device to section a first portion of the imaged tissue in a fluid bath to expose a second region and form a sectioned first portion, transporting the sectioned first portion with a section capture and transport device positioned at the sectioning device to provide a first transported section with individual sections serially indexed and stored in a section storage system, illuminating the second region and detecting light to form an image of the second region, sectioning a second portion in the fluid bath to form a sectioned second portion, transporting the sectioned second portion with the transport system to provide a second transported section so that the first and second transported sections are serially indexed, recording an indexed position for each individual section and for further processing of at least one transported section, and further processing an indexed tissue section to generate tissue section data for each further processed tissue section.
Automated tissue imaging, sectioning and transport with further processing that generates optical tomography and other image types
Illuminating a first region of tissue and detecting light to form a first image of the first region, controlling a sectioning device to section a first portion of the imaged tissue in a fluid bath to expose a second region and form a sectioned first portion, transporting the sectioned first portion with a section capture and transport device to provide a first transported section with individual sections serially indexed for further processing, illuminating the second region and detecting light to form an image of the second region, sectioning a second portion in the fluid bath to form a sectioned second portion, transporting the second sectioned portion with the transport system to provide a second transported section such that the first and second transported sections are serially indexed and an indexed position is recorded for each individual section, and further processing an indexed tissue section to generate a second image of at least one region of tissue comprising an optical tomography image, a multiphoton image, a widefield image, a confocal image or a time resolved fluorescence image.
Across the independent claims, the invention centers on automated imaging of tissue regions followed by fluid-bath sectioning and transport to serially indexed storage, then further processing that generates processed tissue section data and additional image data, with analysis or image generation based on pre-sectioning region images and/or indexed positions.
Stated Advantages
Reduced sectioning artifacts via pre-imaged cut planes.
Accurate registration using overlap-based digital registration/morphing and overlapping volumes.
Automation of whole-organ or thick-tissue section capture with serial indexing and storage for further processing.
Documented Applications
Full mouse heart imaging using the automated workflow, described as taking days versus weeks.
Fast brain atlas generation using the described imaging and section processing workflow.
Multiphasic angiogenesis assay with multi-component readouts including vasculature casting, SHG collagen imaging, nuclei labeling, and hypoxia EF5 imaging.
Immunohistochemistry (IHC) and FISH on captured sections, with processed images indexed and re-imaged for registration back to an original 3D whole-mount dataset.
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