Nanoparticle Tracking Analysis (NTA)
Nanoparticle Tracking Analysis (NTA) is a technique for sizing nanoparticles (such as vesicles) in solution. Size is estimated based upon Brownian Motion (more than you’ll ever want to know about that here). In summary, as light excites small particles, their size can be estimated based on their motion. NTA takes short videos of small particles under magnification following excitation with a laser. These videos are analyzed by software that tracks the motion of individual small particles and creates a histogram of particle sizes in a sample. Concentration can also be estimated using this technique based on known volume of sample in the anlysis chamber.
Limitations for Vesicle Research
- Low throughput (low number of nanoparticles and lengthy setup and data acquisition)
- Non-vesicle specific
- Extremely limited cargo analysis
- Low sensitivity fluorescence is not generally suitable for extremely small number of molecules present on vesicles.
- Inconsistent concentration measurements
- Leads to inconsistent downstream functional studies
Nanoparticle Size and Concentration Determination via NTA
- NanoSight™ LM-10 (Malvern) equipped with a 532 nm laser, sCMOS camera and a syringe pump.
Samples to be analyzed via NTA must be reasonably pure isolations of vesicles of interest.
- Ten videos of 10 seconds each were acquired for each sample
- Using syringe pump, introduce a new volume of sample into the field of view between each acquisition.
- Acquisition settings we generally use: camera level 16, detection threshold 6, with maximum jump length, blur, and min track length all set to Auto.
- Analyze data using NanoSight software (version 3.2).