EVAnalyzer

EVAnalyzer automates quantitative analysis of single extracellular vesicles from fluorescence microscopy images to characterize EV heterogeneity and quantify EV subpopulations.

Key Features:

• Automated Quantification: Detects and quantifies individual extracellular vesicles as light-diffraction-limited fluorescent spots in confocal and widefield fluorescence microscopy images.
• Single-cell–single-vesicle Uptake Pipeline: Implements an automated pipeline for quantifying cell uptake at the single-cell–single-vesicle level suitable for high-content plate-based screening.
• Labelling and Antibody Analysis: Supports validation of (immuno-)labelling reagents and in situ determination of antibody specificity, sensitivity, and species cross-reactivity for EV markers.
• Synthetic Nanoparticle Analysis: Extends analysis to lipid-based nanoparticles (LNPs) and SiO2 nanoparticles to assess siRNA encapsulation efficiency and protein loading.
• Fiji Integration: Implements image-based automated quantitative analysis as a plugin for Fiji.

Scientific Applications:

• Quantification of EV Subpopulations: Enables detailed analysis of different EV subtypes to study heterogeneity and distinct biological roles.
• Validation of Labelling Reagents: Facilitates validation of (immuno-)labelling reagents used in EV research to ensure accurate and specific detection.
• Antibody Specificity Testing: Allows in situ determination of antibody specificity, sensitivity, and species cross-reactivity for various EV markers.
• Genetic Engineering Optimization: Aids optimization of genetic engineering approaches for production and characterization of engineered vesicles.
• Nanoparticle Characterization: Supports assessment of siRNA encapsulation efficiencies and protein loading in LNPs and SiO2 nanoparticles.
• High-Content EV Uptake Assays: Supports automated high-content assays for plate-based single-cell–single-vesicle uptake quantification.

Methodology:

Implemented as a Fiji plugin that detects light-diffraction-limited fluorescent spots and performs automated quantification from confocal and widefield fluorescence microscopy images, including an automated pipeline for single-cell–single-vesicle uptake quantification.