研究目的
To review current and emerging methods for imaging extracellular vesicles (EVs) to facilitate accurate monitoring and study of their spatiotemporal properties in biological and therapeutic contexts.
研究成果
EV imaging is crucial for understanding EV biology and therapeutic applications, but requires careful selection of labeling and imaging methods to ensure accuracy. Future developments should focus on minimizing artifacts and enabling long-term, precise tracking for preclinical and clinical use.
研究不足
The review highlights limitations such as artifacts from sample preparation (e.g., dehydration in EM causing cup-shaped EVs), persistence of dyes outlasting EVs leading to false signals, potential EV fusion or morphology changes from labeling methods like electroporation, and the need for careful consideration of reporter properties to avoid steric hindrance or altered EV function.
1：Experimental Design and Method Selection:
The review discusses various imaging techniques including electron microscopy (TEM, SEM, cryo-EM), atomic force microscopy (AFM), optical microscopy (bioluminescence and fluorescence imaging), and clinical tools (SPECT, PET, MRI), along with labeling strategies such as fluorescent proteins, organic dyes, and radiolabeling.
2：Sample Selection and Data Sources:
EVs are derived from various cell types (e.g., cancer cells, stem cells) and isolated using methods like differential ultracentrifugation.
3：List of Experimental Equipment and Materials:
Includes microscopes (e.g., TEM, SEM, AFM), imaging systems (e.g., IVIS, CCD cameras), dyes (e.g., DiR, PKH67), and contrast agents (e.g., USPIO, 99mTc-HMPAO).
4：Experimental Procedures and Operational Workflow:
Describes sample preparation (fixation, labeling), imaging processes, and in vivo administration routes (e.g., intravenous injection).
5：Data Analysis Methods:
Involves image analysis for biodistribution, quantification of EV properties, and statistical comparisons.
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Transmission Electron Microscope
TEM
Used for imaging the structure of EVs with high resolution.
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Scanning Electron Microscope
SEM
Used for surface topography imaging of EVs.
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Cryo-Electron Microscope
Cryo-EM
Used for imaging EVs in their native hydrated state.
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Atomic Force Microscope
AFM
Used for topographic imaging and quantification of EVs.
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In Vivo Imaging System
IVIS
Used for in vivo imaging of bioluminescent or fluorescent EVs.
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Charge-Coupled Device
CCD
Used for detecting bioluminescence or fluorescence signals.
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Dye
DiR
Lipophilic dye for labeling EV membranes for fluorescence imaging.
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Dye
PKH67
Lipophilic carbocyanine dye for EV labeling.
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Dye
R18
Lipid labeling dye for studying EV fusion with cells.
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Contrast Agent
USPIO
Ultra-small superparamagnetic iron oxide nanoparticles for MRI contrast in EV imaging.
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Radioisotope
99mTc-HMPAO
Used for SPECT imaging of EVs.
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Ultracentrifuge
UC
Used for isolating EVs from cell culture media.
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