Multiplexed Fluorophore Imaging via Mutual Information Minimization
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Solution Overview
Problem
Current multiplexed imaging techniques are limited by the ability to simultaneously image only up to four fluorophores due to spectral overlap, requiring lengthy multi-round staining processes and complex calibration procedures, which hinder efficient imaging of diverse biomolecules in biological samples.
Innovation Solution
The method involves selecting fluorophores with overlapping emission spectra and using mutual information minimization to unmix signals, allowing for simultaneous imaging of up to 10 fluorophores with improved accuracy and reduced imaging time, eliminating the need for spectral detectors and complex calibration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional spectral unmixing methods are used to image multiple fluorophores, then imaging accuracy is maintained, but the number of fluorophores that can be simultaneously imaged is limited to four due to spectral overlap
Solution Approach 1:
The patent changes the parameter of fluorophore selection from requiring spectrally distinct fluorophores to using fluorophores with similar emission spectra. This parameter change enables simultaneous imaging of up to 10 fluorophores by using mutual information minimization to resolve spectral overlaps, thereby increasing the quantity of fluorophores without sacrificing unmixing accuracy.
2Quantity of substance
If multi-round staining processes are used to image diverse biomolecules, then comprehensive biomolecule coverage is achieved, but imaging time increases significantly
Solution Approach 1:
The patent segments the imaging process into a single-round simultaneous imaging approach, dividing the sample into multiple fluorophore channels that are imaged concurrently. This eliminates the need for sequential multi-round staining, reducing imaging time while maintaining the ability to image diverse biomolecules through the use of up to 10 spectrally-similar fluorophores.
3Measurement precision
If spectral detectors and complex calibration procedures are used, then unmixing accuracy is improved, but device complexity and calibration requirements increase
Solution Approach 1:
The patent extracts the spectral unmixing computation from the hardware level to the software level. Instead of using complex spectral detectors and calibration procedures, the method uses standard detectors with a computational approach based on mutual information minimization. This removes the need for expensive spectral detectors and complex calibration while maintaining high unmixing accuracy.
4Measurement precision
If image registration is performed for multi-round staining, then spatial alignment is achieved, but processing complexity and potential errors increase
Solution Approach 1:
The patent performs preliminary action by capturing all fluorophore signals in a single imaging round with spatially registered detectors. This eliminates the need for subsequent image registration processing, as all images are already spatially aligned from the outset. The mutual information minimization is performed on the already-registered images, simplifying the overall processing pipeline.
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 significantly enhances image unmixing accuracy, reduces imaging time, and enables three-dimensional multiplexed imaging without the need for spectral detectors, making it suitable for various biological samples and clinical applications.
Implementation Method 1
The fluorophores are excited by absorbing lights and then emit lights, and at this time, it emits lights with a longer wavelength than the absorbed lights
Data Source
AI summary
Multiplexed fluorescent imaging which is essential for finding out how various biomolecules are spatially distributed in cells or tissues is disclosed. The present disclosure may obtain 10 or more different biomolecule images with one labeling and imaging by newly designing selection of fluorophores, detection spectral ranges, and signal unmixing algorithm. The present disclosure is a blind unmixing technology for unmixing an image without an emission spectrum of fluorophore, and in this technology, 4 pairs of fluorophores are used, and each pair consists of two fluorophores in which emission spectra are overlapped. Each pair of fluorophores is strongly excited by only one excitation laser. Two images with different detection spectral ranges are obtained for each pair, and two images are unmixed via mutual information minimization without fluorophore emission spectrum information. Two images also may be unmixed via Gram-Schmidt orthogonalization and fluorescence measurement based unmixing. This signal unmixing is repeated for each pair of fluorophores. Furthermore, a total of 10 or more fluorophores may be simultaneously used by adding two large stoke's shift fluorophores emitting light in wavelength ranges that does not overlap with the emission spectra of the above 8 fluorophores.