Serial Blockface Staining for Deep Tissue Imaging
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Solution Overview
Problem
Current methods for imaging and staining whole organs and 3D tissues are inefficient, as traditional optical microscopy struggles to image deeply into opaque tissues, and existing techniques face challenges with dye penetration and distribution, leading to labor-intensive and time-consuming processes that are not suitable for high-throughput applications.
Innovation Solution
The implementation of serial blockface staining (SBS) systems and methods that integrate automated cycles of staining, 3D surface and subsurface imaging, and sectioning, allowing for rapid diffusion of stains into exposed tissue surfaces during imaging and sectioning, utilizing multiphoton microscopy and controlled diffusion devices to enhance penetration and distribution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If traditional optical microscopy is used to image tissues, then the imaging process is simple, but the imaging depth is limited to a few tens of microns due to optical opacity
Solution Approach 1:
The tissue is mechanically sectioned into thin slices (ranging from tens of nanometers to hundreds of microns thick) using a microtome or other sectioning device. This segmentation allows optical microscopy to image each thin section individually, overcoming the limited penetration depth into opaque tissues while maintaining simple optical imaging techniques.
2Quantity of substance
If staining reagents are applied to whole organs, then the staining process is straightforward, but the reagents cannot penetrate deeply and evenly into tissues due to diffusion limitations and steric constraints
Solution Approach 1:
The tissue is sectioned into thin slices, which dramatically increases the surface area to volume ratio. This segmentation allows staining reagents to penetrate evenly throughout the tissue depth in a reasonable time, overcoming the diffusion limitations and steric constraints that prevent uniform staining of whole organs.
Solution Approach 2:
The staining process is applied in the lateral dimensions across multiple thin sections rather than attempting to penetrate deeply in the vertical dimension through the entire organ. This dimensional approach allows uniform staining distribution by treating many thin surfaces simultaneously.
3Illumination intensity
If serial section analysis is performed to label and image thick tissues, then deep tissue imaging is achieved, but the process is costly and massively labor intensive requiring days to weeks
Solution Approach 1:
Multiple functions are merged into a single integrated system: the microtome sections tissues and immediately delivers sections to a staining chamber, while an automated slide scanner images sections in real-time. This consolidation of sectioning, staining, and imaging operations into one automated workflow eliminates manual handling and dramatically increases throughput compared to traditional serial section analysis.
Solution Approach 2:
The system performs automated sectioning, staining, and imaging without requiring skilled histologist intervention for each step. The microtome automatically sections tissue, the staining chamber automatically applies and processes stains, and the slide scanner automatically captures and processes images, enabling high-throughput operation.
4Ease of operation
If sections are removed from the tissue block for staining and imaging, then the imaging process is simplified, but irreducible artifacts are introduced from the sectioning process
Solution Approach 1:
Instead of removing and manipulating physical tissue sections, the system uses optical copying methods (light microscopy, confocal microscopy, or two-photon microscopy) to create digital images of the tissue sections in situ. This copying approach allows imaging without physical handling that introduces artifacts, while still providing detailed visual information.
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
This approach enables rapid and even staining of entire organs, reducing imaging time significantly, allowing for high-throughput processing of multiple samples with improved image quality and overcoming limitations of traditional methods by facilitating deep tissue imaging and staining.
Implementation Method 1
Diffusion, due to its random walk nature, is a poor transport mechanism to distribute biochemical labels over large tissue volumes
Implementation Method 2
Other imaging methods such as light sheet, confocal or two-photon microscopy could be used to image the tissue block at tens of microns below the surface
Data Source
Figure 1
Figure 2A~2C
Figure 2D
AI summary
The present invention relates to systems and methods for sequential operation of staining, imaging and sectioning of tissue samples by a processing system. After each layer of the sample is removed by the sectioning system, the system automatically stains the exposed surface of a sample to a depth to enable imaging of the remaining tissue. The system then repeats the sectioning, staining and imaging steps in sequence to image the sample.