Fluorescent Array Grid Alignment and Segmentation
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Solution Overview
Problem
Conventional methods for producing and analyzing nucleic acid and protein arrays face challenges such as low accuracy, high costs, and the need for specialized optical scanning instrumentation, particularly in high-throughput applications, and are limited in producing cDNA and protein arrays with rapid customization.
Innovation Solution
The use of chemically encoded microparticles (beads) assembled into planar arrays through light-controlled electrokinetic assembly or direct disposition, allowing for 'instant' multicolor imaging and automated analysis of receptor-ligand interactions without confocal laser scanning, with methods for grid alignment, segmentation, and clustering in fluorescence images.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If confocal laser scanning is used for imaging arrays, then measurement precision is improved, but productivity deteriorates due to slow scanning speed
Solution Approach 1:
The patent replaces the mechanical confocal laser scanning system with a digital imaging system using standard optical microscopes and CCD/CMOS sensors. This substitution eliminates the slow mechanical scanning process while maintaining sufficient imaging precision through direct digital capture of the entire array in a single snapshot or rapid sequential captures.
Solution Approach 2:
The patent employs periodic illumination with different wavelength lights to excite different fluorescent dyes sequentially. By rapidly alternating between different light wavelengths and capturing images at each phase, the system achieves multicolor imaging without requiring slow sequential scanning, thereby improving productivity while maintaining measurement precision.
2Manufacturing precision
If in-situ photochemical synthesis is used for producing probe arrays, then manufacturing precision is improved for short oligonucleotides, but adaptability deteriorates due to redesign requirements
Solution Approach 1:
The patent segments the probe synthesis process by pre-synthesizing oligonucleotide probes in solution phase with high precision, then separately assembling them onto the array substrate. This separation allows the synthesis step to be optimized for precision while the assembly step provides flexibility for rapid customization and rearrangement of probes on different arrays.
Solution Approach 2:
The patent performs preliminary synthesis of oligonucleotide probes in solution before array fabrication. This preliminary action allows probes to be synthesized with high precision using established solution-phase chemistry, and then stored or rapidly assembled onto arrays as needed, providing both manufacturing precision and adaptability for different array configurations.
3Productivity
If spotted arrays are used for high-throughput testing, then productivity is improved, but measurement precision deteriorates due to accuracy demands
Solution Approach 1:
The patent replaces the spotting mechanical process with a self-assembly or drop-casting method for array fabrication, followed by high-precision fluorescent imaging using digital sensors. This substitution maintains the high throughput capability while improving measurement precision through better signal detection and reduced mechanical variability in probe deposition.
Solution Approach 2:
The patent employs multiple fluorescent dyes with distinct emission spectra to encode different probes or targets on the same array. By using colorimetric detection with spectral unmixing algorithms, the system achieves high measurement precision for distinguishing multiple targets simultaneously, thereby maintaining both high throughput and accuracy.
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 enhances the confidence and efficiency of assay data analysis by enabling accurate grid alignment, segmentation, and clustering of signal sources in fluorescence images, overcoming limitations of conventional methods and allowing for rapid and reliable analysis of large volumes of samples.
Implementation Method 1
Microparticle arrays displaying oligonucleotides or proteins of interest can be assembled by light-controlled electrokinetic assembly
Implementation Method 2
Capture of target or ligand to particular capture agents displayed on carriers of corresponding type as identified by a color code produces an optical signature such as a fluorescence signal
Data Source
AI summary
Disclosed are methods of processing images, and in particular aligning and orienting grids from a fluorescent array of signal sources, by linking nearest neighbor sources to form a hexagon, and then using the hexagon lines for alignment and orientation of the grid. Also disclosed are methods and algorithms for aligning image with grid and correcting for signal beads which are smaller than a grid field and the shift in a grid field. These methods can be used where the signal sources are fluorescent images from a microarray. Also disclosed are methods of automated watershed clustering following transformation.