Single-Cell Fluorescence Calibration for Autofluorescence Correction
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Solution Overview
Problem
Flow cytometry methods suffer from high background signals and autofluorescence, leading to measurement errors and reduced sensitivity in detecting target molecules within cells, particularly due to unspecific binding of markers and variations in staining processes, which result in false negatives and positives.
Innovation Solution
A method involving immobilizing cells on a solid substrate, determining their position, measuring autofluorescence, and then contacting them with detection reagents to bind target molecules, allowing for individual calibration of fluorescence signals by subtracting background values from each cell's autofluorescence, enabling multiple cycles of detection for improved sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry is used to detect target molecules in cells, then cell analysis can be performed, but background signals and autofluorescence cause measurement errors and reduced sensitivity
Solution Approach 1:
The patent measures autofluorescence and background signals before the actual detection step. By performing this preliminary measurement on the same cells before adding detection reagents, the system establishes a baseline for each cell's natural fluorescence. This allows subsequent subtraction of the background signal from the total signal, thereby isolating the specific detection signal and improving measurement precision while eliminating the harmful background interference.
2Measurement precision
If control samples are used to eliminate background signal, then background correction is achieved, but measurement errors occur due to variations between cell populations
Solution Approach 1:
Instead of using a single control sample that represents an average population, the patent segments the correction process at the individual cell level. Each cell serves as its own control by having its autofluorescence measured before detection reagent addition. This cell-by-cell segmentation eliminates the problem of population variations affecting the correction, as each cell's specific background characteristics are accounted for individually, thereby improving both background correction accuracy and measurement reliability.
3Productivity
If single measurement is performed per cell, then analysis is simple, but sensitivity is reduced due to inability to calibrate individual signals
Solution Approach 1:
The patent implements multiple measurement cycles for each cell, measuring autofluorescence before detection reagent addition and then measuring again after reagent addition. This continuous measurement approach on the same immobilized cell allows for signal calibration by comparing the two measurements. The useful action of measuring the target molecule continues across multiple time points, enabling accurate calibration while maintaining high productivity through automated sequential measurements.
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 significantly reduces measurement errors by calibrating fluorescence signals on a cell-by-cell basis, enhancing sensitivity and reliability in detecting target molecules, allowing for more accurate analysis of cellular markers and profiles.
Implementation Method 1
measuring the auto-fluorescence of the individual immobilized cells
Implementation Method 2
contacting the immobilized cells with a first detection reagent comprising (a) a moiety that specifically recognizes and binds a first target molecule
Implementation Method 3
measuring the fluorescence of the fluorescent label of the detection reagent bound to the first target molecule
Data Source
AI summary
Methods for single cell analysis by determining the presence and/or amount of one or more target molecules in a plurality of cells may include: (i) immobilizing said plurality of cells on a solid substrate, wherein the cells are immobilized in form of a monolayer; (ii) determining the position of the individual immobilized cells on the solid substrate; (iii) measuring the auto-fluorescence of the individual immobilized cells; (iv) contacting the immobilized cells with a first detection reagent comprising (a) a moiety that specifically recognizes and binds a first target molecule and (b) a fluorescent label under conditions that allow binding of the detection reagent to the first target molecule; (v) measuring the fluorescence of the fluorescent label of the detection reagent bound to the first target molecule for the individual immobilized cells; (vi) determining the presence and/or amount of the first target molecule in the individual immobilized cells by comparing the fluorescence measured in step (v) with the fluorescence measured in step (iii) on a cell-by-cell basis.