Flow Cell Well Imaging Resolution via Z-Stacking
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Solution Overview
Problem
Conventional optical systems and image processing techniques struggle to achieve desired resolution when increasing the density of wells in a flow cell, particularly due to the challenges of minimizing the distance between adjacent wells while maintaining imaging quality.
Innovation Solution
The system employs a flow cell with multiple reaction sites at different elevations, utilizing a set of cameras and a processor to capture unfiltered images and derive images by removing signals from out-of-focus reaction sites, thereby enhancing imaging resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the density of wells in a flow cell is increased to maximize the number of reactions, then the number of reactions that can be performed is improved, but the imaging resolution deteriorates due to the reduced distance between adjacent wells
Solution Approach 1:
The patent applies focus stacking by capturing images at multiple focal planes (different z-heights) to resolve the contradiction. By imaging reaction sites at different depths separately and combining them computationally, the system achieves high resolution for densely packed wells that would otherwise be indistinguishable in a single-plane image, thus maintaining both high well density and imaging resolution.
Solution Approach 2:
The patent segments the imaging process into multiple focal plane captures, where each capture focuses on a specific depth range. This segmentation allows the system to resolve individual reaction sites at different z-heights separately, preventing signal overlap and maintaining measurement precision even when wells are densely packed in the xy-plane.
2Productivity
If the distance between adjacent wells is minimized to increase well density, then the productivity is improved, but the difficulty of detecting and measuring signals from individual wells increases
Solution Approach 1:
The patent introduces the z-dimension (focal depth) as an additional separation dimension. By capturing images at multiple focal planes and selectively combining signals based on focus criteria, the system can distinguish between closely spaced wells in the xy-plane that would otherwise have overlapping signals, thereby reducing the difficulty of detection and measurement.
Solution Approach 2:
The patent uses computational image processing as an intermediary to separate and identify signals from closely spaced wells. The processing algorithm analyzes focus characteristics across multiple images to determine which reaction site each signal originates from, enabling accurate detection even when physical distance between wells is minimized.
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 allows for improved resolution of reaction sites, enabling the detection of signals from closely spaced wells beyond the diffraction limit, thereby increasing the density of reactions that can be effectively imaged.
Implementation Method 1
an optical system is used to direct an excitation light onto fluorescently-labeled analytes and to also detect the fluorescent signals that may be emitted from the analytes
Implementation Method 2
Each reaction site comprised by that set of reaction sites has a location on that set of reaction sites' corresponding imaging plane
Data Source
AI summary
Resolution of images used in processes such as sequencing by synthesis may be increased by structuring sites that would emit signals in the images to have different elevations. Differences in focus caused by these differences in elevation may be used to filter out background illumination, thereby providing an image in which in focus sites may be resolved even though the separation between any site and its nearest neighbor may be below the diffraction limit of the light that would be emitted.