Flow Cell Alignment Using Dark Field Bead Constellations
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Solution Overview
Problem
Current sequencing instruments using movable flow cells face challenges in accurately aligning and correcting for linear and angular offsets during repeated imaging sessions, leading to misalignment and difficulties in correlating data across successive imaging steps, especially when dealing with small-scale sequencing operations.
Innovation Solution
The implementation of imagers with an XY stage and camera systems that capture dark field images to identify bead constellations, apply correction factors for linear and angular offsets, and adjust tile reference locations to ensure precise alignment and accurate intensity value reading in flow cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If movable flow cells are used for small-scale sequencing operations, then reagent costs and processing costs are reduced, but alignment accuracy and data correlation across imaging sessions deteriorate due to linear and angular offsets
Solution Approach 1:
The system performs preliminary alignment by capturing dark field images of reference beads before main imaging sessions. These reference bead positions are stored and used to calculate correction factors for subsequent imaging sessions, preventing alignment errors before they affect data correlation
Solution Approach 2:
The system continuously monitors bead positions across imaging sessions and uses this feedback to calculate and apply correction factors for linear and angular offsets. This closed-loop approach maintains alignment accuracy despite flow cell movement
2Reliability
If reference locations are adjusted to correct linear offsets, then alignment between imaging sessions improves, but system complexity increases due to additional correction calculations
Solution Approach 1:
The system creates a reference map of bead positions from dark field images that serves as a template for alignment. This reference copy is used to compare against subsequent imaging sessions, simplifying the correction process by providing a stable reference framework
Solution Approach 2:
The system changes reference location parameters dynamically based on calculated offsets. By adjusting reference location coordinates rather than physical hardware, the system corrects alignment errors through software-based parameter modification
3Measurement precision
If dark field imaging is used to identify bead constellations, then alignment precision improves, but imaging time and processing complexity increase
Solution Approach 1:
The imaging process is segmented into distinct phases: dark field imaging for reference bead identification, followed by main imaging sessions. This segmentation allows the system to use simplified dark field imaging only when needed for alignment, rather than continuously
Solution Approach 2:
The system performs partial imaging by capturing only dark field images of reference beads rather than complete high-resolution images of entire flow cells. This partial action provides sufficient alignment information without the time cost of full imaging
Data Source
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AI summary
Imagers and alignment methods for use by imagers imaging deoxyribonucleic acid (DNA) fragments on a flow cell are disclosed. The imagers capture intensity values at DNA fragment bead locations in tiles with each tile having a reference location in the flow cell. Flow cells may be aligned by obtaining a dark field image of each tile during a first imaging session, identifying dark field constellations of bead locations within two separate tiles during the first imaging session, identifying corresponding constellations during a second imaging session, altering the reference location of at least one tile during the second imaging session to correct for a linear offset in the corresponding constellations, and applying at least one correction factor for reading out intensity values from the imager for the bead locations in the flow cell to correct for an angular offset determined from offsets in the corresponding constellations.