Sparse Codebook mFISH Imaging Uniform Negative Control Distribution
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Solution Overview
Problem
Current mFISH imaging techniques face inefficiencies due to inconsistent bit position degeneracy in randomly generated negative control code words, leading to decreased assay throughput, increased reagent use, and reduced data confidence.
Innovation Solution
A method is introduced to generate negative control code words with a uniform distribution of on-values across all code word positions, maintaining a consistent Hamming distance threshold, which reduces false-positive matches and enhances signal confidence for gene identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If randomly generated negative control code words are used in mFISH imaging, then the codebook can be constructed quickly, but bit position degeneracy occurs leading to false-positive matches and reduced data confidence
Solution Approach 1:
The patent changes the generation parameters of negative control code words from completely random assignment to a constrained random assignment where each code word position has an equal probability of being on or off. This parameter change eliminates bit position degeneracy while maintaining the randomness needed for effective negative controls, thereby resolving the contradiction between quick construction and high reliability
Solution Approach 2:
The patent introduces asymmetry in the codebook structure by ensuring that negative control code words have a uniform distribution of on-values across all positions, while gene-specific code words have variable on-value distributions. This asymmetric design allows the system to distinguish true gene signals from false positives effectively, improving data confidence without sacrificing construction efficiency
2Reliability
If more negative control code words are added to the codebook to reduce false-positives, then data confidence improves, but the codebook size increases
Solution Approach 1:
The patent changes the quality parameter of negative control code words from random generation to uniformly distributed generation. This parameter change increases the effectiveness of each individual negative control code word, allowing the system to achieve the same false-positive reduction with fewer code words, thus improving reliability without proportionally increasing codebook size
3Reliability
If uniform distribution of on-values is enforced in negative control code words, then false-positive matches are reduced, but codebook generation complexity increases
Solution Approach 1:
The patent changes the generation parameter from completely random to uniformly distributed random assignment. This parameter change achieves false-positive reduction through a relatively simple algorithmic constraint that ensures equal probability for each bit position, balancing reliability improvement with acceptable generation complexity
Data Source
AI summary
A method of method of spatial transcriptomics includes receiving a plurality of images of a sample from an mFISH imaging system and generating a pixel word represented by a sequence of N intensity values. For each pixel, the pixel word is compared to a codebook and a closest matching code word of a plurality of code words is identified. Each code word is represented by a sequence of N bits. The plurality of code words include a plurality of gene-identifying code words and a plurality of negative control code words, the plurality of negative control code words have an equal number of on-values, and on-values of the plurality of negative control code words are evenly distributed across the N bits such that each ordinal position in the sequence of N bits has a same total number of on-bits from the plurality of negative control code words.