Automated Microtome and Two-Photon Microscopy for Tissue Imaging
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Solution Overview
Problem
Current imaging techniques, such as two-photon microscopy, face limitations in depth penetration and field of view, making it impractical for imaging whole organs or thick tissues with high resolution and speed, and traditional methods like immunohistochemistry struggle with staining whole mount tissues due to diffusion issues and mechanical distortions during sectioning.
Innovation Solution
Integration of an automated microtome into a high-speed two-photon microscopy system for alternating optical and mechanical sectioning, allowing for rapid imaging of thick tissues and subsequent processing of sections for further biochemical analysis, combined with a robotic stage for precise registration and imaging of larger fields of view.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If two-photon microscopy is used for imaging thick tissues, then subcellular resolution and molecular specificity are achieved, but penetration depth is limited to less than a millimeter
Solution Approach 1:
The tissue sample is mechanically sectioned into multiple thin slices using a microtome, with each section being imaged separately by two-photon microscopy. The sections are then digitally reconstructed to form a complete 3D image of the entire tissue volume, overcoming the penetration depth limitation while maintaining subcellular resolution.
Solution Approach 2:
The patent replaces the limitation of optical penetration depth with a mechanical sectioning system (microtome) that physically divides the thick tissue into thinner sections, allowing light to penetrate each section fully while maintaining high resolution imaging capabilities.
2Length of stationary object
If mechanical sectioning is used to prepare tissue sections, then deeper imaging becomes possible, but tissue structures undergo stretching, compressing, and rotation causing alignment difficulty
Solution Approach 1:
Fiducial markers (such as holes or altered tissue features) are embedded in the tissue before sectioning. These markers are copied across all sections and serve as reference points for digital alignment and registration, allowing accurate reconstruction of the original tissue architecture despite mechanical distortions.
Solution Approach 2:
The imaging system detects the positions of fiducial markers in each section and uses this information to dynamically adjust alignment parameters during digital reconstruction, compensating for stretching, compressing, and rotation effects through iterative feedback correction.
3Area of stationary object
If whole organs are imaged using current techniques, then comprehensive coverage is achieved, but imaging speed and data throughput are insufficient
Solution Approach 1:
The large tissue sample is divided into multiple smaller sections that can be imaged in parallel or rapid sequence. This segmentation allows the imaging system to process smaller volumes at high speed while maintaining comprehensive coverage through digital assembly of all sections.
Solution Approach 2:
The system uses rapid alternating cycles of mechanical sectioning and optical imaging, with automated stage movement and focus adjustment between sections. This periodic operation enables high-throughput processing of entire organs by continuously cycling through section preparation and imaging phases.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables high-resolution, three-dimensional imaging of whole organs with improved depth penetration and speed, allowing for accurate registration and analysis of tissue sections, overcoming limitations in data acquisition and mechanical distortion, and facilitating comprehensive biochemical analysis.
Implementation Method 1
two-photon microscopy (TPM) is particularly promising. TPM is a fluorescent optical microscopy technique
Implementation Method 2
an automated microtome integrated into a high speed TPM system. By alternating and overlapping optical sectioning with mechanical sectioning
Data Source
AI summary
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.