Single Cell Western Blot for Live-Lysed Data Correlation
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Solution Overview
Problem
Current methods cannot correlate information from live cells with that from the same cells after lysis, requiring separate analysis of live and lysed cells, which increases the number of cells needed and experimental repetitions while not allowing for matching of data between the two states.
Innovation Solution
A method for multi-layer analysis of single cells using single cell Western blot (scWestern) that involves live cell imaging, cell lysis, electrophoretic separation of cell content, staining, and associating live cell data with proteome data at a single cell resolution, enabling the correlation of live and lysed cell information from the same sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If separate analysis of live and lysed cells is performed, then complete information on both states can be obtained, but the number of cells and experimental repetitions increases
Solution Approach 1:
The patent merges the analysis of live cells and lysed cells into a single integrated workflow. Live cell imaging is performed first, then cells are lysed in the same well without transfer, allowing both live and lysed cell data to be obtained from the same physical sample and well location, thereby eliminating the need for separate analyses and reducing the number of cells required
Solution Approach 2:
The patent performs live cell imaging and data acquisition before lysis occurs. By capturing live cell information (fluorescent signals, cell morphology, etc.) before the lysis step, the system preserves the ability to correlate live state data with subsequent lysed cell data from the same cell, enabling comprehensive analysis without requiring additional cells
2Loss of information
If separate analysis of live and lysed cells is performed, then complete information on both states can be obtained, but experimental repetitions increase
Solution Approach 1:
The patent combines multiple analytical steps into a single integrated experiment. By performing live cell imaging, lysis, and proteome analysis all within the same well without requiring cell transfer or separate experimental setups, the system eliminates redundant experimental repetitions and improves overall experimental efficiency while maintaining complete data correlation
Solution Approach 2:
The patent creates a universal platform where the same sample and well location serve multiple functions: live cell imaging, lysis, and proteome analysis. This multi-functional approach allows a single experimental setup to provide both live and lysed cell data, eliminating the need for separate experiments and reducing computational and resource requirements
3Loss of information
If live cell imaging is performed before lysis, then live cell data can be correlated with lysed cell data, but the workflow complexity increases
Solution Approach 1:
The patent performs live cell imaging as a preliminary step before lysis, capturing all necessary live cell data (fluorescent signals, cell position, morphology) while the cell is still intact. This timing is critical because it allows subsequent lysis to be performed in the same well without losing the ability to correlate live and lysed data, as the imaging data is already acquired and associated with the correct cell identity
Solution Approach 2:
The patent enables the system to serve itself by automatically associating live cell data with lysed cell data through the same well location and cell identity markers. The workflow is designed so that the same physical well and cell serve both imaging and lysis functions, eliminating the need for manual correlation or additional tracking steps
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 reduces the number of cells and experiments required, provides more reliable information by matching data from live and lysed states at a single cell level, and allows for a wider range of cell component analysis, including protein expression and treatment effects.
Implementation Method 1
electrophoretically separating cell content on a gel and immobilizing cell proteome content
Implementation Method 2
Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths
Data Source
AI summary
Improved methods for analyzing cells by combining information from the same cell before and after lysis. The methods of the present invention allow examination of the interactions or relationships between cellular physiological parameter that can only be measured in living cells (i.e., metabolic state) and the expression of proteins and peptides of interest. Single cell processing is made possible by determination of wells containing single cells therein and avoiding analysis of well containing no or multiple cells therein. Fluorescent and/or bright-field imaging may be used in certain embodiments.