Imaging of Extracellular Vesicles by Atomic Force Microscopy
September 11th, 2019
13.6K views
A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.
Overview
This protocol outlines a step-by-step procedure for the label-free immobilization of exosomes and extracellular vesicles from liquid samples, followed by imaging using atomic force microscopy (AFM). The method allows for the estimation of vesicle size and characterization of biophysical properties.
Key Study Components
Area of Science
- Neuroscience
- Biophysics
- Imaging Techniques
Background
- Extracellular vesicles play a crucial role in intercellular communication.
- Atomic force microscopy (AFM) provides high-resolution imaging capabilities.
- Label-free techniques reduce the risk of altering vesicle properties.
- Electrostatic immobilization is a convenient method for sample preparation.
Purpose of Study
- To develop a reliable protocol for imaging extracellular vesicles.
- To compare vesicle sizing results with traditional methods like CryoTEM.
- To facilitate the analysis of vesicle shape and size in different states.
Methods Used
- Isolation of extracellular vesicles from bio-fluids.
- Electrostatic immobilization on surfaces.
- AFM imaging in hydrated and desiccated forms.
- Data analysis to interpret vesicle characteristics.
Main Results
- Vesicle sizing results are consistent with CryoTEM imaging.
- Electrostatic fixation minimizes shape distortion during imaging.
- Hydrated samples require careful handling due to additional factors.
- The protocol is suitable for both new and experienced AFM users.
Conclusions
- The described protocol offers a simple and effective method for vesicle imaging.
- Results can enhance understanding of vesicle biophysics.
- This technique provides a cost-effective alternative to traditional imaging methods.
Frequently Asked Questions
What are extracellular vesicles?
Extracellular vesicles are membrane-bound particles released by cells that play a role in cell communication.
Why use atomic force microscopy for imaging?
AFM provides high-resolution images and can analyze vesicles without the need for labeling.
What is the advantage of label-free techniques?
Label-free techniques prevent alterations to the vesicle properties that can occur with labeling.
How does electrostatic immobilization work?
Electrostatic immobilization uses charge interactions to fix vesicles onto surfaces for imaging.
What challenges are associated with hydrated samples?
Hydrated samples can be affected by environmental factors, requiring careful handling during imaging.
Can this protocol be used for beginners?
Yes, the protocol is designed to be accessible for both new and experienced users of AFM.
Executive Industry Relevance
Atomic force microscopy (AFM) provides a cost-effective, accessible alternative to cryo-TEM for extracellular vesicle (EV) sizing, enabling broader adoption in early discovery workflows. The protocol supports label-free, quantitative characterization of EV size and biophysical properties in both hydrated and desiccated states, with results consistent with gold-standard imaging. This facilitates target validation and mechanistic de-risking by improving confidence in EV-based biomarker and therapeutic hypotheses.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through direct measurement of EV size distribution, supporting biological de-risking of vesicle-mediated mechanisms.
- Operational Value: Provides reproducible, label-free sizing data that reduces variability in early-stage target validation assays.
Screening & Assay Development
- Scientific Value: Generates standardized, quantitative EV size outputs suitable for assay standardization and high-content screening readiness.
- Operational Value: Supports scalable platform reuse across hydrated and desiccated sample formats, improving throughput in EV-based screening campaigns.
Translational & Preclinical Research
- Scientific Value: Ensures translational continuity by providing size data aligned with cryo-TEM, supporting biomarker qualification and preclinical model relevance.
- Operational Value: Enables risk-adjusted advancement decisions through consistent, cross-platform EV characterization.
Pipeline & Workflow Integration
AFM-based EV sizing fits within the discovery continuum from hypothesis testing to lead identification, offering a scalable, quantitative readout that supports go/no-go decisions prior to preclinical investment.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling precise measurement of EV biophysical properties linked to cellular origin and function.
- Screening: Delivers assay-ready, reproducible size distributions that enhance compound evaluation reliability in EV-associated pathways.
- Analytics: Provides dimensional and geometric data from height and phase images, enabling statistical comparison of EV populations under varying conditions.
- Translational Research: Connects discovery to preclinical continuity through size consistency with cryo-TEM, supporting biomarker alignment and disease-relevant system validation.
- Enterprise Reuse: Establishes a reusable, label-free imaging capability applicable across multiple EV isolation workflows and therapeutic areas.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in EV size and shape, reduction of mechanistic ambiguity in vesicle-mediated signaling.
- Operational Value: Standardization, reproducibility, and scalability of EV sizing across hydrated and desiccated states.
- Strategic Value: Better go/no-go decisions, capital efficiency via reduced reliance on cryo-TEM, and lower late-stage biological risk in EV-targeted programs.
- Portfolio Impact: Risk-adjusted prioritization of EV-based biomarkers and therapeutics through validated, cross-platform sizing data.
Implementation Considerations
- Requires expertise in AFM operation, sample preparation, and electrostatic surface modification techniques.
- Depends on access to AFM instrumentation with liquid and air scanning capabilities, along with appropriate cantilevers for hydrated and desiccated imaging.
- Necessitates cross-team standardization of immobilization timing (e.g., 12-hour incubation) to ensure reproducible surface concentration and data quality.
- Involves adaptation considerations for varying EV sources (e.g., MCF7 exosomes) and buffer conditions to maintain vesicle integrity during immobilization and imaging.
- Includes practical limitations such as shape distortion due to electrostatic attraction, requiring post-acquisition correction via tip convolution and surface reconstruction algorithms.
Biopharma FAQs
Why does vesicle sizing matter for target validation in EV-based therapeutics?
Quantitative sizing of extracellular vesicles via AFM supports target validation by enabling precise characterization of vesicle populations linked to cellular origin and function, reducing mechanistic ambiguity in early discovery.
How does electrostatic immobilization improve reproducibility in EV imaging workflows?
Electrostatic immobilization using nickel-modified mica ensures predictable, time-dependent vesicle attachment, allowing consistent surface density and minimizing variability during AFM data acquisition.
What quantitative outputs does AFM provide for extracellular vesicle analysis?
AFM generates height and phase images enabling geometric and dimensional characterization of immobilized vesicles, including size distribution analysis from 561 particles in the study, with results consistent with cryo-TEM.
Why are replication requirements important for AFM-based EV sizing in collaborative projects?
Replication ensures reliable sizing data across users and sites, particularly given the time-dependent surface concentration of immobilized vesicles, which affects scan quality and analytical accuracy.
What statistical analysis is needed before implementing AFM for EV sizing in drug discovery?
Implementation requires tip erosion correction, surface reconstruction, plane leveling, row alignment, and scar removal to ensure accurate vesicle identification and size measurement from AFM data.
