Flow Cytometer Autofluorescence Noise Subtraction
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Solution Overview
Problem
Flow cytometry experiments face challenges in accurately distinguishing between fluorescent markers due to overlapping emission spectra and autofluorescence noise, leading to inaccurate characterization of cell populations.
Innovation Solution
The method involves measuring and associating fluorescence intensity values with forward scatter-side scatter plot regions for both unstained and stained samples, using the forward and side scatter intensity measurements to estimate and subtract autofluorescence noise signals, thereby improving the accuracy of marker detection and reducing noise interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescence intensity values are measured for stained samples, then marker detection capability is improved, but autofluorescence noise interferes with measurement accuracy
Solution Approach 1:
The patent measures fluorescence intensity values for unstained samples before stained sample analysis. These preliminary measurements of autofluorescence noise are then subtracted from the stained sample measurements, allowing the harmful autofluorescence signal to be removed before final analysis, thereby improving marker detection accuracy.
Solution Approach 2:
The patent extracts and removes the autofluorescence noise component from the total fluorescence signal by measuring it separately in unstained samples and subtracting it from stained sample measurements. This extraction isolates the harmful noise signal so it can be eliminated, leaving only the marker-specific fluorescence signal for accurate detection.
2Adaptability or versatility
If multiple fluorescent dyes are used to distinguish different markers, then marker differentiation capability is improved, but overlapping emission spectra cause measurement interference
Solution Approach 1:
The patent applies local quality by measuring and subtracting autofluorescence noise specifically for each fluorescence channel and detector combination. This allows the system to maintain the ability to use multiple fluorescent dyes with overlapping spectra while correcting for noise interference in each specific measurement channel, thereby preserving marker differentiation capability without sacrificing measurement accuracy.
3Measurement precision
If scatter parameters are used to identify cell types, then cell population characterization is improved, but autofluorescence adds noise to the measurements
Solution Approach 1:
The patent converts the harmful autofluorescence noise into a useful correction signal by measuring it in unstained samples. The measured autofluorescence values are then subtracted from stained sample measurements, transforming the noise that initially degraded measurement quality into a correction factor that improves the accuracy of cell population characterization based on scatter parameters.
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 enhances the accuracy of flow cytometry data by effectively compensating for autofluorescence noise and improving the separation of fluorescent peaks, allowing for more precise characterization of cell populations and markers.
Implementation Method 1
particles, such as molecules, analyte-bound beads, or individual cells, in a fluid suspension are passed through a detection region in which the particles are exposed to an excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured
Implementation Method 2
the light scattering and fluorescence properties of the particles are measured. Markers, such as cell surface protein components of cells the presence of which can serve as a distinguishing characteristic, may be recognized by reagents that include fluorescent dyes to facilitate detection, identification, and characterization
Data Source
AI summary
Methods and systems for operating a flow cytometer can include forward scatter values, side scatter values, and fluorescence intensity values for events of an unstained sample and associating the fluorescence intensity values with forward scatter-side scatter side scatter plot regions. Methods and systems for operating a flow cytometer can also include measuring forward scatter values, side scatter values, and fluorescence intensity values for events of a stained sample, determining forward scatter-side scatter plot locations for the events of the stained sample, and for each event of the stained sample, subtracting the fluorescence intensity value associated with the forward scatter-side scatter plot region that contains the forward scatter-side scatter plot location of the stained sample event from the measured fluorescence intensity value of the stained sample event at that forward scatter-side scatter plot location.