Live-Cell Nuclear Translocation Imaging Without Extra Markers
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Solution Overview
Problem
Existing methods for evaluating signaling pathways in cells require cell lysis and time-consuming sample preparation, making them inefficient for real-time monitoring.
Innovation Solution
A method involving capturing non-fluorescence and fluorescence images of live cells with fluorescent protein-based nuclear translocation reporters, using a computational model to identify nuclear pixels and calculate the amount of reporters within and outside nuclei, without the need for separate fluorescent markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If end point assays requiring cell lysis are used to evaluate signaling pathways, then measurement precision is improved, but productivity deteriorates due to time-consuming sample preparation
Solution Approach 1:
The patent replaces mechanical cell lysis and manual sample preparation with automated fluorescence imaging and computational analysis. The system captures fluorescence images of live cells and uses algorithms to automatically quantify signaling pathway activity, eliminating the need for physical cell disruption and manual processing steps.
Solution Approach 2:
The system enables self-service analysis by automatically detecting and quantifying signaling pathway markers in live cells without requiring manual intervention. The computational model processes fluorescence images and generates quantitative measurements of signaling activity directly from the imaging data, allowing the system to perform analysis autonomously.
2Measurement precision
If separate fluorescent markers are used to label nuclei, then measurement precision is improved, but device complexity increases and fluorescent channels are consumed
Solution Approach 1:
The patent employs self-service by having the computational model automatically identify nuclear regions using the fluorescent signal from the signaling pathway marker itself. The system uses algorithms to detect and segment nuclear regions based on the fluorescence distribution, eliminating the need for separate nuclear markers or additional fluorescent channels.
Solution Approach 2:
The fluorescent signal from the signaling pathway marker serves multiple functions: it indicates both the location and activity state of the signaling pathway, and simultaneously provides the information needed to identify nuclear regions. This multi-functional use of a single fluorescent marker reduces the need for additional markers and simplifies the imaging system.
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
Enables efficient, real-time monitoring of signaling pathways by simplifying image analysis and freeing up fluorescent channels for additional cellular component analysis.
Implementation Method 1
capturing a fluorescence image of the fluorescent protein-based nuclear translocation reporters in the one or more live cells
Data Source
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AI summary
A method for monitoring one or more live cells includes capturing a non-fluorescence image of a sample that includes one or more live cells that further contain fluorescent protein- based nuclear translocation reporters (FTRs), capturing a fluorescence image of the FTRs in the live cell(s) in the sample, identifying, via a computational model, nuclear pixels of the non-fluorescence image that correspond to nuclei of the live cell(s), identifying, based on the nuclear pixels, first pixels of the fluorescence image that correspond to the nuclei and second pixels of the fluorescence image that do not correspond to the nuclei, and calculating, based on first intensities of the first pixels and second intensities of the second pixels, a metric representing a first amount of the FTRs located within the nuclei of the live cell(s) and a second amount of the FTRs not located within the nuclei of the live cell(s).