On-Chip Localization Microscopy Substrate for Sequencing Throughput
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Solution Overview
Problem
Current sample analysis technologies, such as DNA sequencing, are limited by low maximum density of sample material that can be handled, restricting throughput and efficiency in genetic research and analysis.
Innovation Solution
An on-chip analysis substrate with a localization layer and a sensor layer, including a spacer layer, that enables single-molecule localization microscopy (SMLM) by receiving fluorescence from fluorescent dyes, allowing for higher sample density imaging and improved sequencing efficiency through precise localization and imaging techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional imaging methods are used, then the system is simple to operate, but the sample density is limited and throughput is low
Solution Approach 1:
The patent transitions from conventional widefield imaging to single-molecule localization microscopy (SMLM), which adds a temporal dimension to the imaging process. By capturing multiple images over time and localizing individual molecules in each frame, the system achieves super-resolution capability that enables higher sample density imaging, directly improving sequencing throughput without requiring physical changes to the sensor array dimensions.
Solution Approach 2:
The patent changes the imaging parameters by using SMLM techniques including stochastic activation of fluorophores, temporal gating, and centroid calculation from point spread functions. These parameter changes allow the system to resolve individual molecules at densities that would be indistinguishable in conventional imaging, effectively increasing the information capacity per sensor pixel and improving throughput.
2Productivity
If sample density is increased, then throughput is improved, but measurement precision becomes more difficult to maintain
Solution Approach 1:
The patent segments the imaging process into multiple temporal frames, where only a sparse subset of fluorophores is activated in each frame. This segmentation in time allows precise localization of individual molecules even at high overall densities, because each frame contains few enough active emitters to maintain measurement precision. The final super-resolution image is reconstructed by combining localizations from all frames.
Solution Approach 2:
The patent employs periodic activation and deactivation of fluorophores through controlled illumination and chemical reactions. This periodic action ensures that at any given time, only a manageable number of fluorophores are emitting, maintaining localization precision while allowing high overall sample density. The cycle repeats across multiple frames to build the complete image.
3Device complexity
If on-chip imaging is implemented, then device integration is improved, but optical path control becomes more difficult
Solution Approach 1:
The patent introduces an intermediary optical path through the use of a flow cell chamber with controlled thickness and refractive index matching. This intermediary structure facilitates efficient fluorescence collection by minimizing optical aberrations and maximizing light transmission from the sample plane to the sensor, making fluorescence detection feasible in the integrated on-chip configuration.
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
Enhances the density of sample material that can be analyzed, enabling multiple clusters to be imaged per sensor pixel and improving sequencing throughput by utilizing SMLM for precise localization and imaging, thereby overcoming the limitations of existing technologies.
Implementation Method 1
a localization layer to be provided with a sample comprising a nucleotide provided with a fluorescent dye; one or more of the array of sensor pixels to receive a propagation of fluorescence from the fluorescent dye
Data Source
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AI summary
An analysis substrate comprises: a localization layer to be provided with a sample comprising a nucleotide provided with a fluorescent dye; and a sensor layer comprising an array of sensor pixels, the localization layer being on-chip relative to the sensor layer, one or more of the array of sensor pixels to receive a propagation of fluorescence from the fluorescent dye.