Nanoparticle Tracking Analysis (NTA) tracks scattered light signals from small particles in the suspension using a microscope equipped with charge-coupled device (CCD) or scientific complementary metal-oxide-semiconductor (sCMOS) camera over multiple frames. Nanoparticles (NPs) in suspension move under Brownian motion. This motion is related to the particle size based on the Stokes-Einstein equation. NTA software adopts this equation to estimate the size distribution and concentration of all types of nanoparticles at 10 nm to 2000 nm in diameter and 106 to 109 particles per mL. For bimodal mixture samples, NTA can distinguish size differences of 25% (1:1.25 ratio). Thus, compared to the more commonly used DLS (Dynamic Light Scattering) technique, NTA is able to measure the hydrodynamic size of NPs with better size resolution in a small concentration range, although the applicable size range is narrower than DLS technique.
NTA combines a microscope and a CCD camera to visualize and record scattered laser light from suspended nanoparticles. The hydrodynamic diameter of particles, bubbles or other liquid droplets dispersed in the suspension is related to the Brownian motion parameter of the Stokes-Einstein equation. NTA software identifies and tracks NPs moving under Brownian motion, and measures particle movement values to determine particle size based on a formula derived from the Stokes-Einstein equation. Since the measured particles are assumed to be completely spherical, the actual size and the measured size become similar if the sample particles are close to the sphere.
The amount of light scattering and the speed of Brownian motion depend on the size and composition of the particles. NTA can measure particle size by measuring the intensity of scattered light and tracking the particle position with Brownian motion. Generally, Brownian motion of small particles is fast and the scattering of large and metal particles is strong. Concentration is measured from the number of particles measured in a specific volume specified by the manufacturer(see Figure 3).
The average spatial displacement of particles per unit time can be calculated by tracking individual particles in arbitrary Brownian motion frame in units of frames. Frames are still images obtained by capturing video of moving particles in NTA device. This displacement is related to the hydrodynamic diameter of the particles in the Stokes-Einstein equation. Brownian motion is a three-dimensional process, but hydrodynamic diameter of a particle can be found using one-, two-, or three-dimensional diffusion coefficients (three equations below).
The above equations are the mean square displacement values of 1D, 2D and 3D, respectively. t is the time between sequential displacement measurements. NTA assumes motion in two dimensions and uses the following equation:
A 20x magnification microscope and camera tracks the Brownian motion of each particle in real time via video. The diffusion coefficient (Dt) is obtained by measuring the mean squared displacement (displacement of particles over time) in two dimensions with particle tracking software.
Stokes Einstein equation is used to determine the size of the hydrodynamic sphere (d). Particle size (d) is calculated, and what NTA actually measures is the speed of particle move. Thus, temperature (T) and viscosity (η) affect the calculated particle size. The number of particles to be measured depends on the sample concentration, scattering volume, particle size (since it affects the diffusion rate) and analysis time. The larger this number, the better the reproducibility of the entire sample. If the size distribution is wide, more particle measurements are required because the number of statistically significant particles in each size class of the distribution must be measured. For monodisperse samples, less than 100 particles are sufficient for measurement.
DLS shows the average value of a sample, so it is good to know the representativeness, but it is difficult to distinguish the characteristics of individual particles. On the other hand, NTA tracks individual particles and provides a statistical distribution of particle sizes, so that sample information does not disappear based on signal strength. NTA can distinguish size differences of 25% (1:1.25 ratio) but DLS can distinguish differences by more than 300% (1:3 ratio).
Generally, reagents related to this technique are not required. However, in order to maintain colloidal stability during measurement, certain samples often require dispersants and stabilizers. Proper dilution is necessary to obtain an appropriate particle concentration for the measurement. As the property or concentration of the ionic environment in which the particles are dispersed changes, the hydrodynamic size or diffusion coefficient of the particles may change.
※ The NanoSight system mainly measures the concentration range of ~107-109 particles/mL, which is the concentration at which 20 ~ 100 particles are observed on the screen. The recommended concentration is about 3X108 particles/mL, with 30-80 particles on the screen.
Turn off the power and remove the power connector from the sample holder. Inject the sample into the chamber of the O-ring Top-Plate using a 1 mL syringe. There should be no air bubbles in the syringe. To avoid air bubbles in the chamber of plate, place the sample inlet downward and slowly inject the sample from the bottom up. After injecting the sample, reconnect the power connector and place it on the microscope. Turn on the laser and connect the thermometer.
Turn on the NTA device first, then run the software.
※ Since Brownian motion is highly affected by the ambient vibration, it should be measured in the absence of ambient vibration.
For more information, see ‘C. Software execution’.
The NS300 has two sample holders:
② The O-Ring Top-Plate can be used to measure samples except fluorescent samples.
LVFC is useful when the amount of sample is small and can be used to measure fluorescent samples. Use DI water-based solvents if possible because it is vulnerable to fat-soluble, organic solvents, and pH.
※ When ijnspecting fluorescent samples, wrap the syringe in aluminum foil to block the light and, if necessary, perform a sample measurement in flow mode using a syringe pump.
Turn on the NTA device first, then run the software. Check the connection between the system and the PC in the software.
For NS300, auto focusing is available. First, use autofocus to focus, then use the fine focus knob to refocus if necessary. The NS300 has a neutral density (ND) filter and fluorescent filters.
※ Notes on Cleaning: Wipe Gently wipe the optical plate part in one direction with lens-cleaning tissues. The syringe used for cleaning should be used only once and discarded to prevent contamination.
You can control the Camera shutter and Camera gain in the Adv Camera tab of the Hardware tab.
Too many particles can affect the movement of the particles, and scattered light from other particles can result in improper measurements. Therefore, the number of particles that can be seen on the screen (field of view) is suitable for 30 to 80.
Too many particles can affect the movement of the particles, and the measurement may not be performed properly due to scattered light from other particles. Therefore, the number of particles visible on the screen (field of view) is 30 to 80 as described in <III Sample Preparation C Precautions>.
Adjust the detection threshold to select the particles to be included in the trace. The detection threshold must be determined by the user to determine whether it is a real particle or not. To check the real particle trace, move the slide at the bottom of the screen to identify particles in the entire video frame and then determine the detection threshold.
NS300 can be measured in flow mode using a syringe pump.
(It is recommended to adjust the particles to disappear from the screen for about 10 seconds. Infusion rate is usually about 50. However, it is better to judge the time by watching the screen. Because the particle's moving speed changes with size).
The NS300 can measure fluorescent samples.
You can change the graph that comes out when you measure
In the Software, select Preference -> Graph. When the graph
settings are displayed, you can set the No. of Bins in the size graph box (maximum value is 2000)