Free-State Fluorescence Compensation for Stable Flow Cytometry
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Solution Overview
Problem
Fluorescent dyes used in flow cytometry are prone to degradation due to environmental factors, leading to fluctuations in fluorescence radiant intensity, which affects the accuracy of blood cell characterization and requires complex handling and additional measurement units.
Innovation Solution
A method for quantifying and compensating for fluorescence radiant intensity variations by measuring the free-state fluorescence of the dye, using a single optical transducer to derive a normalization function for bound-state fluorescence measurements, eliminating the need for additional hardware and complex fluidic systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent dyes are used to enhance blood cell characterization, then measurement precision is improved, but reliability deteriorates due to dye degradation from environmental factors
Solution Approach 1:
The patent implements a feedback mechanism by continuously monitoring the free-state fluorescence intensity of the dye and using this information to adjust bound-state measurements. The system measures the actual fluorescence intensity of free dye molecules in solution and uses this feedback to compensate for degradation effects in real-time, maintaining measurement reliability while preserving the enhanced characterization capability
Solution Approach 2:
The patent applies preliminary action by measuring the free-state fluorescence intensity before performing bound-state measurements. This preliminary measurement establishes a baseline that accounts for any dye degradation or environmental effects, allowing the system to pre-correct subsequent measurements and maintain accuracy throughout the analysis process
2Measurement precision
If additional measurement units are used to monitor dye degradation, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent applies universality by designing a single optical transducer that performs multiple functions: it measures both the free-state fluorescence intensity (to monitor degradation) and the bound-state fluorescence intensity (for cell characterization). This multi-functional approach eliminates the need for separate measurement units, maintaining high measurement precision while avoiding increased device complexity
3Reliability
If complex fluidic systems are used to handle dye degradation, then reliability is improved, but device complexity increases
Solution Approach 1:
The patent replaces complex mechanical fluidic systems with an optical-based solution. Instead of using mechanical means to prevent or compensate for dye degradation, the system uses optical measurements of free-state fluorescence to detect and correct degradation effects. This substitution maintains measurement reliability while dramatically simplifying the overall system architecture by eliminating complex fluidic handling mechanisms
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 method effectively stabilizes the fluorescence intensity of fluorescent dyes, ensuring accurate blood cell characterization by normalizing bound-state fluorescence levels, reducing instrument complexity, and minimizing environmental impact.
Implementation Method 1
fluorescence radiant intensity monitoring of a reagent containing a fluorescent dye used in flow cytometry
Implementation Method 2
fluorescent markers can be chosen to be highly specific of a targeted blood cell feature such as blood cell nucleic acids content. Once excited, i.e illuminated at a specific wavelength, fluorescent probes re-emit fluorescent light
Data Source
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AI summary
The present invention relates to a method of determining a compensation factor to compensate for variations of fluorescence radiant intensity of a reagent containing a fluorochrome, the compensation factor being intended to be applied during a bound state fluorescence radiant intensity measurement when a cell is labeled with the fluorochrome, the method comprising at least the steps of: - measuring a free state fluorescence radiant intensity emitted by the reagent containing the fluorochrome when not labeled to a cell prior to the bound state fluorescence radiant intensity measurement, - determining the compensation factor from the measured free state fluorescence radiant intensity.