Multiplex Fluorescence Imaging via Sequential Marker Sets
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Solution Overview
Problem
Current techniques for analyzing biological samples with fluorescence microscopy can only image a low number of different fluorescent dyes with high spatial resolution, limiting the identification of cell types and the predictive power of results.
Innovation Solution
A method involving multiple sets of markers, each with a unique fluorescent dye and affinity reagent, excited by distinct wavelength spectra, allowing for the generation of multiple images with increased channel capacity without removing or deactivating previous markers, enabling high spatial resolution imaging of a larger number of markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If fluorescence microscopy is used to image multiple fluorescent dyes, then the number of markers that can be imaged increases, but the spatial resolution deteriorates
Solution Approach 1:
The patent divides the imaging process into multiple sequential rounds, where different sets of fluorescent dyes are imaged in separate time periods. Between rounds, the sample is repositioned or the detection system is reconfigured. This temporal segmentation allows high spatial resolution to be maintained for each individual imaging round while accumulating data from many different fluorescent markers across multiple rounds, thereby resolving the contradiction between imaging more markers and maintaining spatial resolution.
2Loss of information
If more fluorescent dyes are imaged simultaneously, then the identification of cell types improves, but the device complexity increases
Solution Approach 1:
The patent employs periodic action by cycling through multiple imaging rounds, where each round focuses on imaging a specific subset of fluorescent dyes with optimized detection parameters. The system periodically reconfigures between rounds, adjusting excitation wavelengths, detection channels, or sample positioning. This periodic approach enables comprehensive cell type identification across many markers while keeping the complexity of any single imaging configuration manageable.
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 increases the number of markers that can be imaged with high spatial resolution, enhancing the identification of cell types and the reliability of results by allowing for the simultaneous visualization of multiple structures within biological samples.
Implementation Method 1
directing first excitation light having a first wavelength spectrum onto the biological sample in order to excite the fluorescent dyes of a first set of markers, and generating at least one first image from fluorescence light emitted by the excited fluorescent dyes
Data Source
AI summary
A method for analyzing a biological sample (1002) comprises: Providing a plurality of markers (1612), each marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) comprising a fluorescent dye (1320) unique to the marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) and an affinity reagent (1310 to 1319) unique to the marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526), the affinity reagent (1310 to 1319) being configured to attach to a predetermined structure (1706 to 1714) within the sample (1002). Staining the sample (1002) by introducing the plurality (1612) of markers into the sample (1002). Directing first excitation light having a first wavelength spectrum onto the sample (1002) in order to excite the fluorescent dyes (1320) of a first set of markers (1614). Generating at least one first image from fluorescence light emitted by the excited dyes of the first set (1614), the first image comprising at least two channels, each channel corresponding to one marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) of the first set of markers (1614). Directing at least one second excitation light having a second wavelength spectrum onto the sample (1002) in order to excite the fluorescent dyes (1320) of a second set of markers (1616), the second set (1616) being distinct from the first set (1614). Generating at least one second image from fluorescence light emitted by the excited dyes of the second set (1616), the second image comprising at least two channels, each channel corresponding to one marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) of the second set of markers (1616).