Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Summary

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Abstract

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Introduction

Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. They contain lipids, proteins, metabolites, and nucleic acids and transfer these materials locally between cells and distally between tissues and organs. There are three primary subtypes of EVs: apoptotic bodies, microvesicles, and exosomes^1,2. Here, we focus our discussion on exosomes and their associated proteins.

Exosomes are secreted vesicles originating from the inward budding of early endosomes into the multivesicular body (MVB). The MVB then fuses with the plasma membrane, releasing the exoso....

Protocol

1 Propagation and maintenance of cell lines

1. Acquire human osteosarcoma cells (U2OS) and place the cells in the growth medium supplemented with 10% exosome-free Fetal Bovine Serum and 1x Penicillin/Streptomycin solution.
   NOTE: Exosome-free Fetal Bovine Serum was generated following the protocol presented in McNamara et. al.²⁶.
2. Maintain the U2OS cells in a copper-coated incubator at 37 °C in 5% CO₂ and passage cells in T175 flasks^26,27. The cells must be maintained in mid-logarithmic growth phase to prevent subpopulations from arising, or from ....

Results

The goal of this study was to evaluate the effectiveness of super-resolution microscopy in visualizing individual EVs with nanometer resolution in three dimensions (3-D). To analyze the shape and size of individual EVs, we employed photoswitchable dye and incubated the EVs with a far-red, membrane intercalating dye, and removed excess dye through chromatography²⁹. The affinity-captured anti-CD81 and red-stained EVs were then viewed in the super-resolution microscope under the 640 nm excitation las.......

Discussion

EVs have become a popular area of study due to their important role in many intracellular processes and cell-to-cell signaling^1,30. However, their visualization proves to be difficult as their small size falls below the diffraction limit of light microscopy. Direct stochastic optical reconstruction microscopy (dSTORM) is a direct method of visualization that bypasses the diffraction limit by capturing photoswitching events of individual fluorophores over time and.......

Materials

Name	Company	Catalog Number	Comments
15 µ-Slide 8 well plates	Ibidi	80827
1X PBS	Gibco	14190-144
1X Penicillin Streptomycin solution	Gibco	15140-122
50 mL conical tube	Thermo Fisher	339652
500 mL 0.22 µm vacuum filtration apparatus	Genesee	25-227
750 kDa hollow-fiber cartridge cutoff filter	Cytiva	29-0142-95
AKTA Flux S	Cytiva	29-0384-37
AKTA Start	Cytiva	29022094-ECOMINSSW
Anti-CD81 magnetic beads	Thermo Fisher	10616D
B-cubed buffer	ONI	BCA0017
CellMask Red	Thermo Fisher	C10046
Dubelco's Modified Eagle Medium	Thermo Fisher	10566016
Fetal Bovine Serum	VWR	97068-085
Frac 30 Fraction collector	Cytiva	29022094-ECOMINSSW
Glycine pH=2.0	Thermo Fisher	BP381-5
HiTrap CaptoCore 700 Column	Cytiva	17548151
Molecular Biology Grade Water	Corning	9820003
Nanoimager	Oxford Nanoimaging	Custom
Paraformaldehhyde	Electron Microscopy Sciences	15710
Polyethylene glycol	Thermo Fisher	BP233-1
RNase A	Promega	A797C
T175 Flasks	Genesee	25-211
Tetraspek microspheres	Invitrogen	T7279
Tris- HCl pH=7.5	Thermo Fisher	BP153-1
Unicorn V	Cytiva	29022094-ECOMINSSW