Fluorophore Spectral Profiling Using Two-Image Reference Unmixing
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Solution Overview
Problem
Fluorescence imaging techniques face challenges due to spectral overlap between fluorophores, leading to complex image clarity issues and sensitivity to noise levels, particularly in multiplex imaging where multiple fluorescent dye markers are used.
Innovation Solution
A method involving the acquisition of two images for each sample, one optimized for individual fluorophores and another for all fluorophores, followed by linear unmixing to determine spectral information and minimize crosstalk, using a predefined unmixing matrix derived from controlled acquisition settings and fluorophore selection to optimize signal-to-noise ratio.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If linear unmixing is used to manage spectral overlap, then image clarity is improved, but sensitivity to noise levels and background autofluorescence increases
Solution Approach 1:
The patent applies preliminary action by acquiring reference spectral information from control samples (single-stained samples) before performing linear unmixing on the multiplexed sample. This reference data is used to pre-calculate the unmixing matrix, which then corrects spectral overlap in the final image. By preparing the unmixing parameters in advance based on controlled reference measurements, the system improves image clarity while reducing sensitivity to noise in the final multiplexed image analysis.
2Loss of information
If multiple fluorescent dye markers are used for multiplex imaging, then information about various targets is increased, but spectral overlap between dyes complicates image clarity
Solution Approach 1:
The patent applies segmentation by dividing the multiplexed sample analysis into separate single-stained control samples. Each control sample contains only one fluorescent dye marker, allowing the spectral characteristics of each individual dye to be measured separately. These segmented measurements are then used to construct an unmixing matrix that can deconvolute the spectral overlap in the final multiplexed image, thereby maintaining image clarity while enabling multiplexed target detection.
Solution Approach 2:
The patent introduces an intermediary element - the unmixing matrix calculated from single-stained control samples - that mediates between the multiple fluorescent dye markers and the final image analysis. This intermediary computational model represents the spectral contributions of individual dyes and enables the separation of overlapping signals in the multiplexed sample, allowing simultaneous detection of multiple targets without spectral overlap complications.
3Measurement precision
If staining is performed in multiple rounds with washing or bleaching, then spectral overlap is reduced, but effort and time required increase considerably
Solution Approach 1:
The patent applies preliminary action by performing single-stained control staining experiments before the final multiplexed imaging. These preliminary staining experiments with individual dyes allow the system to capture reference spectral information that is then used to calculate the unmixing matrix. This approach eliminates the need for time-consuming multiple-round staining with washing or bleaching steps, as the spectral unmixing is achieved computationally based on the preliminary reference data.
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
Enhances image clarity by effectively reducing crosstalk and improving signal-to-noise ratio in multiplex fluorescence imaging, allowing clearer differentiation of fluorophores.
Implementation Method 1
Fluorescence imaging allows for the use of multiple fluorescent dye markers to label various targets within a sample
Data Source
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AI summary
A first aspect of the present disclosure is related to a determination of spectral information of fluorophores in stained samples, comprising the steps: - select a set of fluorophores ; - preparing a set of samples to comprise one or more fluorophores from the set of fluorophores ; - acquiring a first image for each of the prepared samples based on excitation of the fluorophores comprised by the respective sample; - acquiring a second image for each of the prepared samples based on excitation of the fluorophores of all samples and/or of the set of fluorophores ; - determining spectral information based on the two images for one or more predefined spectral channels .