FRET Microscopy with Controlled Fluorophore Activation for Pathogen Detection
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Solution Overview
Problem
Existing molecular detection techniques struggle to selectively identify specific biological pathogens due to low selectivity of pathogen biomarkers, which can distinguish only between general classes of microorganisms and not specific species or strains, and cell metabolites are common to many cell types, making discrimination difficult.
Innovation Solution
A system and method utilizing fluorescence resonance energy transfer (FRET) with controlled donor and acceptor fluorophores, employing photoactivatable or chemically activatable fluorophores to detect molecular structures by identifying FRET events, including single molecule localization microscopy (SMLM) to determine interaction distances and locations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional biomarker detection methods are used, then detection of general microorganism classes is possible, but specific species or strain identification is not achieved
Solution Approach 1:
The detection system segments the detection process into multiple stages: initial FRET-based detection for general class identification, followed by single-molecule FRET analysis for specific species/strain identification. This segmentation allows each method to be optimized for its specific purpose, resolving the contradiction between general detection capability and specific identification precision.
Solution Approach 2:
The patent introduces FRET (Förster Resonance Energy Transfer) as an intermediary mechanism that bridges the gap between general biomarker detection and specific molecular identification. By using donor-acceptor fluorophore pairs as intermediaries, the system achieves high-resolution detection of specific molecular structures without relying on traditional low-selectivity biomarkers.
2Measurement precision
If fluorescent probes are used for molecular structure detection, then FRET events can be detected, but high fluorescence background noise masks the FRET signal
Solution Approach 1:
The patent extracts and isolates the FRET signal from the background noise by using single-molecule detection techniques. Instead of detecting FRET events in a bulk population where fluorescent background dominates, the system identifies and analyzes individual FRET-bearing molecules, effectively separating the signal of interest from the harmful background fluorescence.
Solution Approach 2:
The system creates a copy of the FRET event signal through multiple detection channels and temporal/spectral separation. By recording fluorescence emissions at different wavelengths and times, and using computational algorithms to reconstruct the FRET signal, the system creates a purified copy of the signal that is free from background noise contamination.
3Adaptability or versatility
If multiple fluorophores are used for detection, then detection coverage is improved, but distinguishing between closely spaced fluorophores becomes difficult
Solution Approach 1:
The patent resolves the spatial resolution conflict by adding temporal and spectral dimensions to the detection. Instead of relying solely on spatial separation to distinguish closely spaced fluorophores, the system uses temporal modulation (different excitation/emission timing) and spectral characteristics to differentiate between fluorophores, effectively adding new detection dimensions that resolve spatial ambiguities.
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
Enables accurate detection of specific molecular structures by minimizing background noise and distinguishing between closely spaced fluorophores, allowing precise identification of DNA, RNA sequences, and pathogens at the sub-nanometer scale.
Implementation Method 1
the respective fluorophores provide optical fluorescence in response to exciting illumination. When attached to the target structure, the donor and acceptor fluorophores are at close proximity between them, enabling FRET process between the fluorophores
Implementation Method 2
the fluorophores are provided to the sample in combination with selected molecular probes and are selected such to allow FRET interaction between the donor and acceptor fluorophores at certain range of distances between them
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3A~3B
AI summary
A system and method are presented, for monitoring and/or imaging of a sample. The system comprises: a light unit configured for illuminating the sample in at least two different wavelength ranges; a collection unit configured for collecting a light emitted from the sample in at least a third wavelength range and directing said emitted light towards at least one detector; and an activation unit configured for providing activation signal to selectively activate at least a portion of fluorescent substance in the sample; and a processing circuitry configured for operating the light unit to determine a selected temporal illumination profile of said at least two different wavelength ranges and for operating the activation unit for controllable activation.