Extracellular Vesicle Fixation via Non-Reversible Cross-Linking
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Solution Overview
Problem
Current methods for imaging extracellular vesicles (EVs) in tissues and biological fluids are inefficient, leading to inconsistent results and significant loss of EVs during fixation and imaging processes, hindering the understanding of EV ultrastructure and spatial localization in normal and diseased states.
Innovation Solution
A method using a non-reversible cross-linking agent, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to fix EVs, which prevents their loss and allows for robust transmission electron microscopy (TEM) imaging, enabling visualization of EVs in various biological fluids and tissues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional fixation methods are used to image extracellular vesicles, then imaging can be performed, but significant loss of EVs occurs during fixation and imaging processes
Solution Approach 1:
The patent applies preliminary action by treating EVs with a non-reversible cross-linking agent (such as EDC) before imaging to permanently attach them to the support film. This pre-fixation step prevents EV loss during subsequent imaging procedures, directly resolving the contradiction between enabling imaging and preventing substance loss.
2Measurement precision
If conventional fixation methods are used, then imaging can proceed, but results are inconsistent and EV ultrastructure cannot be properly understood
Solution Approach 1:
The patent changes the chemical parameter of the fixation process by using a non-reversible cross-linking agent instead of conventional reversible fixatives. This parameter change ensures permanent attachment of EVs to the support film, producing consistent and reliable imaging results that reveal true EV ultrastructure, thereby resolving the contradiction between measurement precision and reliability.
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 method significantly improves the retention and visualization of EVs, allowing for the observation of morphological differences and spatial localization, particularly in cancer tissues, and provides a diagnostic tool for diseases mediated by EVs.
Implementation Method 1
contacting the sample with a non-reversible cross-linking agent under conditions effective to fix the extracellular vesicles
Data Source
Figure 1a~1d
Figure 1e~1f
Figure 2a~2c
AI summary
The present invention relates to a method of fixing extracellular vesicles. The method includes providing a sample containing extracellular vesicles and contacting the sample with a non-reversible cross-linking agent and, optionally, an aldehyde-containing fixative. Preferably the non-reversible cross-linking agent is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The method also includes imaging the fixed extracellular vesicles for the determination or exclusion of disease or disorder in a clinical sample. The present invention also relates to a kit for fixing extracellular vesicles in a biological sample.