NTA results are very prone to operator bias. This protocol demonstrates the effects of altered NTA parameters on obtained results. A standardized method will help increase rigor and reproducibility in NTA analysis.
Analyzing the sample in a cuvette allows for a statistically random sample to be captured in each video. This results in more reproducible data and the visualization of particles over a wide range of sizes. As getting a sample in the recommended particle concentration range can be difficult, be sure to perform a serial dilution to identify the ideal dilution factor.
Demonstrating the procedure will be Kungheng Cai, a PhD student from Anthony Ferrante's laboratory. To prepare cuvette for nanoparticle tracking analysis, cover the workspace with a lint-free material to prevent fibers from entering the cuvettes. Wearing gloves, place a cuvette containing a stir bar onto the magnetic cuvette jig.
Use a hook tool to place the insert into the cuvette with the notch of the insert visible at the front of the cuvette. Use a pipette to slowly add 400 to 500 microliters of purified extracellular vesicles onto the cuvette through the hole in the insert and mix the sample by gently pipetting without introducing air bubbles, then cap the cuvette, tapping out bubbles as necessary and use a lint-free cloth to wipe the outside surface of the cuvette. To analyze the particle concentration of the diluent or a sample, turn on the computer workstation and instrument and start the particle tracking analysis program.
When prompted, click NTA and open the recording tab. Follow the onscreen instructions to fill out all the necessary sample information. For EV tracking analysis, set the diluent to PBS.
The salinity will auto-populate to 9%To obtain the diluent or sample particle concentration, open the instrument lid and remove the protective cap covering where the cuvette will be placed. Load the cuvette into the instrument in the correct orientation with the notch of the insert facing the camera and replace the cap and instrument lid. Click the streaming arrow to turn on the camera and click the chevron arrow to expand the record settings.
Adjust the focus until the relatively small particles are clearly visible. To set the analysis for a small EV quantification, set the frame rate to 30 frames per second, the exposure to 15 milliseconds, the stir time to five seconds, the wait time to three seconds, the blue, green, and red laser powers to 210, 12, and 8 milliwatts respectively, the frames per video to 300, and the gain to 30 decibels. Adjust the focus until the relatively small particles are clearly visible.
Increasing the zoom and/or the gain can help with particle focusing. But if you increase the gain, remember to set it to 30 decibels prior to recording. Once particles are in focus, set the zoom setting to 0.5X to save bandwidth and to prevent loss frames and click record to begin recording the video.
When a prompt appears stating that the videos have been recorded, click OK to complete the recording and select the process tab. If very large particles were visible in any video while recording, navigate to the directory of recorded videos and remove any problematic video prior to processing. Check the disable audio detection override box and set the feature diameter to 30.
Click process to initiate video processing and view a live distribution graph. When the processing is complete, click OK and select the plot tab. For EVs, display the main chart as log bin silica.
Other features of the graph, such as changing the x-axis to set the area for integration of the figure produced, can be customized. To create a PDF report of the results, click the report button. The mean, median, mode size, and concentration adjusted for the dilution factor and distribution width will be displayed.
NTA of the diluent should be performed before any sample so that the concentration of this blank can be subtracted from the EV sample particle concentration. To clean the cuvettes after analysis, empty the cuvette and completely fill the cuvettes 10 to 15 times with deionized water and the three times with 70 to 100%ethanol to remove any residual sample. Dry the outside of the cuvettes with a lint-free microfiber cloth and dry the inside with a compressed air duster.
To clean the inserts and stir bars, place the materials in a glass scintillation vial containing 70 to 100%ethanol and shake the vial vigorously. Then rinse the inserts and stir bars in deionized water with shaking as demonstrated and dry them using lint-free cloths. After drying, immediately place all of the clean components into storage until the next analysis.
Before performing an analysis, the instrument calibration was tested using polystyrene beads to ensure the validity of the acquired data. As observed, the particle tracking instrument accurately reported the size of the 100 nanometer manodisperse beads, but only closely reported the size of the 400 nanometer beads. Therefore, the instrument settings for this protocol were more accurate for smaller particles, closer to 100 nanometers in size.
Using these settings, reported particle concentration scales accordingly with the dilution factor demonstrating that the instrument can accurately detect the particle concentration at various dilutions with little variability between technical replicants. The optimum dilution for a 4.41 times 10 to the 10th particles per milliliter mouse tissue-derived EV sample was determined to be between 1, 000 and 3, 000. In this analysis, increasing the gain increased the sensitivity of the camera, allowing an increase in the visualization of a higher number of smaller particles.
Increasing the blue laser power from 70 to 210 milliwatts while keeping the green and red laser powers constant shifted the reported average particle size from 122 to 105 nanometers and increased the reported total particle concentration from 1.1 times 10 to the eighth to 1.7 times 10 to the eighth. Increasing the power of the red laser increased the reported average particle size from 175 to 246 nanometers and decreased the reported total particle concentration. Increasing the green laser power resulted in a decrease in the reported average particle size and an increase in reported total particle concentration.
Finding the right dilution to place a sample within the optimal detection range can take a few tries for each sample. The cuvette cleaning also requires extra careful handling. We recommend applying more than one orthogonal method for EV particle size and concentration measurements.
Dynamic light scattering, resistive pulse sensing, transmission electron microscopy, and single-particle interferometric reflectance imaging sensing can also be performed to characterize EVs.
