Multispectral Image Cube Analysis for Fluorescent Dye Separation
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Solution Overview
Problem
Fluorescence imaging of biological samples is complicated by overlapping excitation and emission spectra of multiple dyes and endogenous fluorescence, making it difficult to distinguish and quantify specific fluorescent dyes in multispectral images.
Innovation Solution
A method and system for calculating a pure spectrum of a fluorescent dye by identifying pixel intensity values in an image cube, separating contributions from the dye and endogenous fluorescence, and using these spectra to determine the relative amounts of the dye in a sample, even when the spectra overlap significantly.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple fluorescent dyes are used to visualize multiple proteins, then the ability to detect multiple antigens is improved, but the difficulty to distinguish and quantify specific dyes increases due to spectral overlapping
Solution Approach 1:
The patent segments the spectral measurement process by acquiring images at multiple discrete excitation and emission wavelength combinations, creating a multispectral image cube. This segmentation allows computational separation of overlapping spectral contributions through systematic analysis of intensity values at different wavelength pairs, enabling distinction between dyes with overlapping spectra.
Solution Approach 2:
The patent introduces endogenous fluorescence as an intermediary reference signal. By measuring the endogenous fluorescence signal at the same excitation wavelengths (where it emits but does not absorb), the system creates a mediator that can be mathematically subtracted from the total signal to isolate the contribution of exogenous fluorescent dyes, thereby enabling accurate quantification despite spectral overlap.
2Reliability
If endogenous fluorescence is present in the sample, then the natural fluorescence emission is captured, but the complexity to determine dye contributions increases
Solution Approach 1:
The patent performs preliminary measurement of endogenous fluorescence by acquiring images at excitation wavelengths where the fluorescent dyes do not absorb (dark bands for the dyes). This preliminary action captures the endogenous fluorescence signal in isolation, allowing it to be stored as a reference and subsequently subtracted from total signal measurements to determine dye contributions without increasing operational complexity.
Solution Approach 2:
The patent converts the complicating factor of endogenous fluorescence into a beneficial reference signal. By measuring endogenous fluorescence at wavelengths where dyes are dark (non-absorbing), the system transforms what was previously a confounding variable into a useful intermediary that enables mathematical separation of endogenous and exogenous signals, simplifying the determination of dye contributions.
3Measurement precision
If spectral unmixing is performed to separate dye contributions, then the accuracy of dye quantification is improved, but the computational complexity increases
Solution Approach 1:
The patent segments the computational task into systematic steps: (1) acquiring intensity values at multiple excitation-emission wavelength combinations, (2) identifying endogenous fluorescence contributions through dark band measurements, (3) subtracting endogenous signal from total signal, and (4) calculating pure dye spectra. This segmentation transforms complex spectral unmixing into a series of manageable computational operations, improving accuracy without excessive complexity.
Solution Approach 2:
The patent creates a computational model by copying the spectral response characteristics of endogenous fluorescence and using this copy to subtract from measured signals. By measuring endogenous fluorescence at reference wavelengths and constructing a spectral copy, the system enables accurate dye quantification through mathematical manipulation rather than complex real-time spectral analysis.
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 allows for accurate determination of fluorescent dye spectra independent of autofluorescence, reducing operator dependence and measurement errors, and is applicable to samples with multiple overlapping dyes, enhancing the reliability and reproducibility of fluorescence imaging analysis.
Implementation Method 1
Fluorescence imaging of a dye involves exciting it with light of a first wavelength band or range of wavelengths, and observing light that it emits in response to this, in a second wavelength band or range of wavelengths
Implementation Method 2
Many samples exhibit endogenous fluorescence emission. That means that when optically excited, the sample itself emits fluorescent light
Data Source
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AI summary
The disclosure features methods that include obtaining multispectral image information for a sample that includes a fluorescent dye, calculating from an image cube of the information a first spectrum and a second spectrum, and calculating a pure spectrum of the fluorescent dye in the sample based on the first and second spectra, where a relative contribution of light emission from the fluorescent dye to the second spectrum is larger than a relative contribution of light emission from the fluorescent dye to the first spectrum, where calculating the first and second spectra includes identifying corresponding first and second sets of pixel intensity values in the image cube, and where identifying the first set of pixel intensity values includes designating one or more layers of the image cube as a first layer set.