Flow Cytometry Signal Normalization Across Detector Gain Settings
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Solution Overview
Problem
Flow cytometers face challenges in maintaining consistent day-to-day performance due to variations in detector gain settings, which affect signal-to-noise ratio and mean fluorescence intensity, making it difficult to optimize both simultaneously.
Innovation Solution
Implementing methods for gain-independent normalization of data signals by adjusting photodetector settings using a scaling factor, which accounts for variations in particle velocity and laser intensity, and employing a calibration factor to maintain consistent mean fluorescence intensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If detector gain settings are adjusted to maximize signal-to-noise ratio, then signal-to-noise performance is improved, but mean fluorescence intensity consistency over time deteriorates
Solution Approach 1:
The patent applies parameter changes by introducing a scaling factor that transforms detector gain settings into gain-independent data. This scaling factor dynamically adjusts the relationship between detector gain and measured signal, allowing the system to maintain both high signal-to-noise ratio and consistent mean fluorescence intensity across different gain settings and over time.
Solution Approach 2:
The scaling factor acts as an intermediary between the detector gain settings and the measured fluorescence intensity. By applying this intermediary transformation, the system decouples the direct relationship between gain settings and signal output, enabling independent optimization of both signal-to-noise ratio and intensity consistency.
2Stability of the object's composition
If detector gain settings are adjusted to ensure consistent mean fluorescence intensity, then day-to-day performance consistency is improved, but signal-to-noise ratio deteriorates
Solution Approach 1:
The patent transforms the measurement approach by introducing gain-independent scaling that changes how data is processed. Instead of adjusting gain settings to control mean fluorescence intensity, the system applies a scaling factor to the data signals, thereby maintaining consistent intensity without compromising signal-to-noise ratio.
Solution Approach 2:
The patent replaces the mechanical adjustment of detector gain settings with a computational scaling factor applied after data acquisition. This substitution allows the system to achieve consistent mean fluorescence intensity through software processing rather than hardware adjustment, preserving the optimal signal-to-noise ratio obtained during data collection.
3Productivity
If detector gain settings are optimized for specific applications, then application-specific performance is improved, but cross-instrument comparability deteriorates
Solution Approach 1:
The patent achieves universality by creating gain-independent data through the scaling factor transformation. This transformed data format maintains the optimized performance characteristics for specific applications while simultaneously enabling consistent cross-instrument comparison, as the scaling factor normalizes the data across different instruments and gain settings.
Solution Approach 2:
The patent applies parameter changes by transforming the data representation through gain-independent scaling. This transformation preserves application-specific optimization by maintaining the relative signal relationships while enabling cross-instrument comparability through standardized normalization, allowing the same analysis protocols to be applied across different flow cytometers.
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
Ensures consistent and calibrated data analysis across instruments and over time, enhancing signal-to-noise ratio and maintaining resolution performance, facilitating efficient cross-instrument analysis and calibration-free consistency.
Implementation Method 1
detecting light from a particle in a sample in a flow stream with a light detection system having a photodetector
Implementation Method 2
The flow stream is irradiated with light. Variations in the materials in the flow stream, such as morphologies or the presence of fluorescent labels, may cause variations in the observed light
Implementation Method 3
Variations in the materials in the flow stream, such as morphologies or the presence of fluorescent labels, may cause variations in the observed light
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
Aspects of the present disclosure include methods for normalization of gainindependent analyte data (e.g., flow cytometer data). Methods according to certain embodiments include detecting light from a particle in a sample in a flow stream with a light detection system having a photodetector, generating data signals in response to the detected light, normalizing the data signal with a detector gain to generate gain-normalized data signals and adjusting the gain-normalized data signals with a scaling factor to generate scaled data signals. Systems and non-transitory computer-readable storage media configured to carry out the subject methods are also provided.