Method Article

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

DOI:

10.3791/62845

⸱

August 26th, 2021

 ,  , 
1Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill School of Medicine

In This Article

Summary

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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

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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

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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 exosomes1,2. 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 exosomes into the extracellular space to travel to other cells3,4. Exosomes exist on a spectrum of sizes ranging from 40 to 150 nm and are enriched with endosomal transmembrane proteins known as tetraspanins (CD9, CD63, CD81), membrane-bound endosomal sorting complex required for the transport (ESCRT), and lipid raft-associated proteins1,2, 5,6,7.

Characterizing the biochemical makeup of exosomes has become a popular field for researchers to better understand their functional nature. Many methods exist for visualizing and characterizing exosomes, including nanoscale flow cytometry, nanoparticle tracking analysis (NTA), scanning and transmission electron microscopy (TEM), surface plasmon resonance, resistive pulse sensing, and traditional light microscopy, each of which contains intrinsic pros and cons8,9. TEM and cryo-EM can achieve nanometer-based resolution, but often require dehydrating and freeze-fracture steps, thereby shrinking or lysing EVs10,11. NTA relies on light scattering, allowing for the characterization of hundreds of EVs at a time, but is an indirect measurement of particle size and cannot easily distinguish between EVs, viruses, and protein aggregates12,13,14,15,16. Nanoscale flow cytometry employs light scattering from an excitation path, which can then be translated into size measurements, but is an emerging technology, and there is little consensus on what size of particles are within the linear range of detection for various instruments12,17,18.

Traditional light microscopy using fluorescent proteins or dyes has been one of the most heavily employed techniques for visualizing subcellular compartments, protein complexes, and signaling machinery within a cell. While this technique proves useful in visualizing the localization of complexes, the diffraction limit of traditional light microscopy (around 250-400 nm) prevents the clear resolution of proteins or structures in the typical size range of an exosome (40-150 nm)12,19,20.

Super-resolution microscopy, namely, direct stochastic optical reconstruction microscopy (dSTORM), distinguishes itself from conventional light microscopy by employing the photoswitchable properties of specific fluorophores and detecting these blinking events to reconstruct images down to nanometer precision21. Photoswitching events are collected using a high-framerate detection camera over the course of tens of thousands of individual exposures, and a point spread function is used to map with high confidence the exact location of the photoswitching fluorophore19,20,22. This allows dSTORM to bypass the diffraction limit of light microscopy. Several groups have reported the use of super-resolution techniques for visualizing and tracking exosomes and their associated proteins22,23,24,25. The final resolution depends on the biophysical properties of the fluorophore, but often ranges from +/-10-100 nm along the XY-axis, allowing single-molecule resolution.

The ability to resolve individual fluorophores at this scale on the XY-axis has revolutionized microscopy. However, there is little data on the three-dimensional (3-D) dSTORM of an exosome. Therefore, we sought to establish a standard operating procedure (SOP) for dSTORM-based visualization and characterization of purified EVs, including exosomes to nanometer precision in 3-D.

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Protocol

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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 flasks26,27. The cells must be maintained in mid-logarithmic growth phase to prevent subpopulations from arising, or from the accumulation of apoptotic debris during the stationary phase.

2 Exosome isolation and purification

  1. Grow the U2OS cell lines to full confluency in 10 separate T175 flasks with 50 mL of media per flask. Remove the cell supernatant and successively passage it through a 0.45 µm and 0.22 µm vacuum filtration apparatus.
  2. Subject the supernatant to crossflow filtration (also known as tangential flow filtration) on a filtration system equipped with the 750 kDa hollow-fiber cartridge in order to remove smaller proteins or metabolites28.
    1. Passage the supernatant through the tangential flow filtration operator at a constant forward pressure of 30 psi, while maintaining a retention pressure of <20 psi to produce a Δ pressure of 10 or more psi. Place a magnetic stir bar in the retentate tank and set to 150 rpm26.
    2. Maintain the feed rate at 40 mL/min or higher. Collect the permeate in a reservoir and dispose of it.
  3. Concentrate the supernatant to 30 mL, and then equilibrate with four volumes of 1x PBS. Collect the crossflow filtered and equilibrated solution in a 50 mL conical tube.
  4. Precipitate the EVs at 4 °C overnight in a final concentration of 40 mg/mL polyethylene glycol with a molecular weight of 8,000 Da. Centrifuge the EVs at 1,200 x g at 4 °C for 1 h and resuspend in 500 µL of 1x PBS.
  5. Incubate the EVs with 50 µg/mL of photoswitchable red membrane intercalating dye and 50 µg/mL of RNase A in 1x PBS at 4 °C for 1 h.
  6. Remove excess dye through a column on a protein purification system attached to a fraction collector. Identify EV fractions through UV absorbance and pool them into a microcentrifuge tube26.
  7. Affinity-select a total of 200 µL of EVs using anti-CD81 magnetic beads equilibrated in 1x PBS.
    1. Dilute 100 µL of the anti-CD81 magnetic beads in 1x PBS to a total volume of 1 mL and allow them to bind at 4 °C for 2 h in a 2 mL microcentrifuge tube with continuous rocking.
  8. Wash the anti-CD81 beads and EV three times with 1x PBS. Elute CD81+ EV from the solution with 100 mM Glycine at pH 2.0 for 30 min at 37 °C.
  9. Pipette the purified CD81+ EV into an equal volume of 100 mM Tris-HCl at pH 7.5 in 1x PBS.
    ​NOTE: An aliquot of purified CD81+ EV solution may be reserved for Nanoparticle Tracking Analysis or electron microscopy in order to confirm the purity of the solution and presence of EVs26.

3 Fixation and preparation

  1. Place affinity-purified EVs onto glass-bottom µ-slide 8-well plates in a total volume of 200 µL (can dilute EV sample in 100 mM Tris-HCl at pH 7.5 in 1x PBS) and allow to adhere to the surface overnight at 4 °C.
  2. Without removing the existing solution from the 8-well plate, fix EVs onto the plates by adding 200 µL of 4% paraformaldehyde in 1x PBS to the EV-containing solution in each well and allow to incubate for 30 min at room temperature21.
  3. Carefully remove paraformaldehyde and excess solution with a micropipette in order to not disturb the EVs. Wash the EV with 1x PBS to remove excess paraformaldehyde. Perform the wash procedure three times. Remove excess 1x PBS.
  4. Prepare 250 µL of dSTORM B-cubed buffer solution per sample by creating a solution of 5 mM protocatechuate dioxygenase (Part A) diluted in imaging buffer (Part B) per manufacturer's protocol.
    NOTE: The concentration of the enzyme in the buffer may be doubled to 10 mM if photobleaching occurs.
    1. Add 250 µL of the prepared buffer to each well per manufacturer's protocol for 20 min at room temperature before imaging to scavenge oxidizing molecules.
      ​NOTE: The EV can then be either viewed immediately or stored at 4 °C for up to a week. Replace the buffer following storage before every visualization.

4 Direct stochastic optical reconstruction microscopy calibration

  1. Prepare the beads required for the calibration of the super-resolution microscope by diluting 100 nm microspheres to a concentration of 0.5% in molecular biology grade water and pipetting 200 µL into each well of a glass-bottom µ-slide 8-well plate.
    1. Allow the beads to settle in the wells for 1 h at room temperature.
  2. Without removing the existing solution, add 200 µL of 4% paraformaldehyde in PBS to each well to the calibration bead solution and allow to incubate for 30 min at room temperature.
  3. Carefully remove the paraformaldehyde with a micropipette to not disturb the beads and wash the beads three times with 1x PBS. Prepare the buffer according to step 3.4.
  4. Remove 1x PBS and add 250 µL of the prepared buffer to each well. Allow the buffer to sit for 20 min before visualization.
  5. Before placing anything on the stage, connect to the 3-D microscope using the Connect the Microscope button. Add 100x oil to the objective and place the center of the well on top of the objective. In the Acquire setting, turn on the 473 and 640 nm excitation lasers and click on View.
    1. Without the 3-D lens activated, view the beads under the photon saturation setting by clicking on Photon Counts in the Image Display options. Set the initial laser powers to 8.4 mW for the 473 nm laser and to 11.6 mW for the 640 nm laser.
    2. Decrease the focus of the laser to around -300 nm or the focal plane of the calibration beads to produce a clear resolution of the individual beads. Once the Z-plane is focused, further adjust the laser power levels to account for variation in each field of view.
  6. Under the instrument functions, complete 3-D mapping calibration and channel mapping calibration to obtain the errors on the X-, Y-, and Z-axis. Set the max number of FOVs to 20, the target number of points to 4,000, the max distance between channels to 5.0 pixels, and the exclusion radius between channels to 10.0 pixels during channel mapping calibration.
  7. Ensure that the calibration produces a point coverage of >90% and mapping quality that is good. Save the given calibration data for future image acquisitions.

5 Visualization of EV in three dimensions

  1. Add 100x oil onto the objective and place the prepared EVs into the microscope. Without the 3-D lens activated, turn on the 640 nm excitation laser and initially raise it to between 1.2 mW and 12.5 mW depending on the intensity of the signal and field of view to excite the red membrane intercalating dye stained EVs.
    1. Under the Image Display options, switch the viewing method from photon saturation to percentiles to better visualize the EVs. Adjust laser power to minimize noise, while maximizing signal. Maintain all the other parameters.
    2. Adjust the focus of the Z-plane by clicking the up or down icon on the Z-axis.
      NOTE: The Z-plane should be focused between -200 and -350 nm, but will vary depending on the field of view.
  2. Set the exposure time to 20 ms, the frame capture to 10,000 frames, and the initial laser power to between 1.2 mW and 12.5 mW, or the laser power determined in step 5.1, depending on the intensity of the signal and field of view.
    1. Activate the 3-D lens using the icon and start the acquisition by clicking on the Acquire button.
  3. Throughout image acquisition, raise the laser power by 3 increments of 10 every 1000 frames, or enough to maintain a high signal-to-noise ratio. Do not adjust the Z-plane during acquisition.
    ​NOTE: The laser may be raised to a maximum of 90 mW laser power.

6 Post-acquisition modification and EV tracing

  1. After the image acquisition, toggle over to the Analyze viewing window. Perform drift correction on the unfiltered image, and then activate filters. Adjust photon count, localization precision, sigmas, and frame index according to Table 1.
  2. Overlay an XYZ plane view tool along the X-axis of individual EVs from the field of view and export .CSV files of photoswitching events.
  3. Bisect individual EVs on the XY-axis in an X by Y field of view using a line histogram tool, which bins photoswitching events into set distance groups.
  4. Take images of single EVs and save them as .tiff files.
  5. Create 3-D videos of individual EVs using a 3-D visualization tool and color according to placement along the Z-axis.

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Results

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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...

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Discussion

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EVs have become a popular area of study due to their important role in many intracellular processes and cell-to-cell signaling1,30. 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...

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Disclosures

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M.G.C. has no conflicts of interest to declare. R.P.M and D.P.D. receive material support from Oxford Nanoimaging (ONI) Inc. and Cytiva Inc. (formerly GE Healthcare). R.P.M. and D.P.D. declare competing interests for the possible commercialization of some of the information presented. These are managed by the University of North Carolina. The funding sources were not involved in the interpretations or writing of this manuscript.

Acknowledgements

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We would like to thank Oxford Nanoimaging for their constructive feedback and guidance. This work was funded by the 5UM1CA121947-10 to R.P.M. and the 1R01DA040394 to D.P.D.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
15 µ-Slide 8 well platesIbidi80827
1X PBSGibco14190-144
1X Penicillin Streptomycin solutionGibco15140-122
50 mL conical tubeThermo Fisher339652
500 mL 0.22 µm vacuum filtration apparatusGenesee25-227
750 kDa hollow-fiber cartridge cutoff filterCytiva29-0142-95
AKTA Flux SCytiva29-0384-37
AKTA StartCytiva29022094-ECOMINSSW
Anti-CD81 magnetic beadsThermo Fisher10616D
B-cubed bufferONI BCA0017
CellMask RedThermo FisherC10046
Dubelco's Modified Eagle MediumThermo Fisher10566016
Fetal Bovine SerumVWR97068-085
Frac 30 Fraction collectorCytiva29022094-ECOMINSSW
Glycine pH=2.0Thermo FisherBP381-5
HiTrap CaptoCore 700 ColumnCytiva17548151
Molecular Biology Grade WaterCorning9820003
NanoimagerOxford NanoimagingCustom
ParaformaldehhydeElectron Microscopy Sciences15710
Polyethylene glycolThermo FisherBP233-1
RNase APromegaA797C
T175 FlasksGenesee25-211
Tetraspek microspheresInvitrogenT7279
Tris- HCl pH=7.5Thermo FisherBP153-1
Unicorn VCytiva29022094-ECOMINSSW

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Tags

Extracellular VesiclesSuper Resolution MicroscopyDirect Stochastic Optical ReconstructionThree Dimensional ImagingdSTORM MicroscopyExosome VisualizationVirus Particle ImagingPhotoswitching EventsSize Distribution AnalysisCalibration Beads

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