Extracellular Vesicle SMLM Characterization with Substrate Immobilization
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Solution Overview
Problem
Current methods for characterizing extracellular vesicles (EVs) are limited in providing a comprehensive and accurate picture at the single-EV level, often requiring multiple technologies due to low throughput, inconsistency, and inability to distinguish between low copy numbers and zero copies of biomarkers, leading to unreliable results.
Innovation Solution
A method involving sample preparation with fluorescently labeled EVs on a substrate, followed by single molecule localization microscopy (SMLM) to determine morphological and copy number parameters, and dimensionality reduction of feature vectors for comprehensive characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional fluorescence microscopy is used to characterize EVs, then the method is relatively straightforward to perform, but it cannot distinguish between low copy numbers and zero copies of biomarkers, leading to unreliable results
Solution Approach 1:
The patent segments the fluorescence signal into discrete single-molecule localizations, allowing individual biomarker molecules to be counted. This segmentation enables distinction between zero copies and low copy numbers by detecting individual fluorescent events, thereby improving measurement precision while maintaining operational simplicity through automated image analysis
Solution Approach 2:
The patent replaces conventional diffraction-limited optical detection with single-molecule localization microscopy (SMLM) that achieves super-resolution by temporally and spatially separating molecular events. This substitution of the detection mechanism enables precise counting of individual biomarker molecules, resolving the contradiction between ease of operation and measurement precision
2Reliability
If multiple complementary analyses are performed to confirm results, then reliability improves, but device complexity and time requirements increase
Solution Approach 1:
The patent creates a universal single-molecule characterization platform that simultaneously provides multiple EV parameters (size, biomarker copy number, morphology) in a single assay. This multi-functional approach achieves reliable comprehensive characterization without requiring multiple separate technologies, thereby reducing device complexity while maintaining high reliability
Solution Approach 2:
The patent merges size determination (via scattering) and biomarker detection (via fluorescence SMLM) into a single integrated assay performed on the same EV population. This combination eliminates the need for separate complementary analyses, reducing both device complexity and analysis time while maintaining reliability through orthogonal measurement within one platform
3Reliability
If EVs are pre-isolated and purified before analysis, then interference from non-EV contaminants is avoided, but significant loss of EVs occurs and morphological damage may result
Solution Approach 1:
The patent introduces substrate immobilization as an intermediary step that captures EVs in a controlled manner, allowing direct analysis of captured EVs without extensive pre-purification. This intermediary approach maintains EV integrity and quantity while enabling specific capture and analysis, achieving reliable results without significant EV loss or morphological damage
4Reliability
If ultracentrifugation is used for EV isolation, then EVs can be separated from contaminants, but the procedure is biased towards EVs of specific size and density
Solution Approach 1:
The patent changes the isolation parameter from density-based ultracentrifugation to surface-based specific capture (e.g., antibody-antigen, lectin-carbohydrate). This parameter change enables capture of diverse EV populations regardless of size or density, improving adaptability while maintaining reliability through specific molecular recognition
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 differentiation of EV populations and characterization of individual EVs, including morphological and compositional features, with high confidence in biomarker detection, reducing variability and improving throughput.
Implementation Method 1
the vesicles are labelled with one or more fluorescent probes; imaging said one or more fluorescent probes on the vesicles
Data Source
AI summary
The present application discloses methods for characterising vesicles. The method involves (1) a sample preparation step, comprising providing a test specimen with vesicles attached to a substrate, wherein the vesicles are labelled with one or more fluorescent probes; (2) an image acquisition step, comprising imaging said one or more fluorescent probes on the vesicles to generate image data; (3) an image processing step which identifies individual vesicles and constructs a feature vector containing characterising parameters for individual vesicles characterising parameters (including a morphological parameter) (4) a data transformation step to calculate modified feature vectors of lower dimensionality for individual vesicles; and (5) a characterisation step, which characterises the vesicles based on the modified feature vectors. The application also discloses methods for immobilising vesicles on a substrate, as well as substrates functionalised to capture vesicles.