Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) - a Comparison
NanoSight have developed a unique instrument which allows the tracking of the Brownian motion of nanoparticles in liquid suspension on a particle-by-particle basis. Subsequent application of the Stokes-Einstein equation allows the determination of particle size. Particle count is also available. This technique presents a powerful alternative to more typical light scattering techniques such as Dynamic Light Scattering (DLS) - otherwise known as Photon Correlation Spectroscopy (PCS), for the analysis of complex and polydisperse sample types of varying composition. This note outlines the main differences in results, technical specifications and system requirements for each technique.
How Do They Work?
Both DLS and NTA measure the Brownian motion of nanoparticles whose speed of motion, or diffusion coefficient, Dt, is related to particle size through the Stokes-Einstein equation.
In NTA this motion is analysed by video - individual particle positional changes are tracked in two dimensions from which the particle diffusion is determined. Knowing Dt, the particle hydrodynamic diameter can be then determined.
Figure 1. Typical image produced by NTA showing particle tracks.
In contrast, DLS does not visualise the particles individually but analyses, using a digital correlator, the time dependent scattering intensity fluctuations. These fluctuations are caused by interference effects arising from the relative Brownian movements of an ensemble of a large number of particles within a sample. Through analysis of the resultant exponential autocorrelation function, average particle size can be calculated as well as a polydispersity index. For multi-exponential autocorrelation functions arising from polydisperse samples, deconvolution can furnish limited information about the particle size distribution profile.
NTA is Ideal for Polydispersed Samples
For the analysis of polydispersed samples (i.e. containing a range of particle sizes) or those which contain different particle types of differing refractive index, the NTA approach is far better suited due to its particle-by-particle measurement. Because DLS is an ensemble measurement which is significantly biased to larger, higher scattering particles, the resulting intensity weighted average can be seriously misleading in the analysis of polydisperse samples.
In a recently published comparison of NTA and DLS, Filipe et al (2010)* stated that “NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS”.
They concluded that “NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.”
In Figure 2 below, comparisons between NTA and DLS measurements are made on bimodal samples of mixtures of different sized particles.
Furthermore, because NTA tracks particles within a known volume of liquid, the size distribution which is produced is a true and direct number frequency distribution and relative particle concentrations can be determined by user-selection of different parts of the distribution.
In contrast, DLS produces an intensity distribution from which, assuming a fixed relationship between scattering intensity and volume, a volume distribution can then be estimated. Applying further assumption about particle shape (a sphere is assumed), a number distribution can be calculated. However, it is well recognised that any intensity weighting errors in the original conversion are compounded and therefore number distributions as calculated through DLS are generally considered to be inaccurate.
*Filipe, Hawe and Jiskoot (2010) “Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates”, Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2
Figure 2. Size distribution from NTA and DLS measurements of mixtures of monodisperse polystyrene beads (middle column) with the corresponding NTA video frame (left column) and 3D graph (size vs. intensity vs. concentration; right column). a) 60-nm/100-nm beads at a 4:1 number ratio; b) 100-nm/200-nm beads at a 1:1 number ratio; c) 200-nm/400-nm beads at a 2:1 number ratio; d) 400-nm/1,000-nm beads at a 1:1 number ratio. (from Filipe et al (2010))
NTA Insensitivity to Contaminants
In the following example, also from Filipe et al (2010), the insensitivity of NTA to the presence of contaminants is shown. While present in the scattering volume, the effect they have on the ability of NTA to both see and analyse the smaller particle population is minimal, although high numbers of larger particles may act to occlude smaller particles behind them resulting in a slight loss of accuracy in estimating the number concentration of smaller particles.
Figure 3. Influence of large particles (1,000-nm beads) in a mixture of 100-nm and 400-nm monodisperse beads on NTA and DLS measurements. The size distribution (middle columns) with the corresponding NTA video frame (left columns) and normalized 3D graph (size vs. intensity vs. concentration; right columns) are shown. a) no 1,000-nm beads; b) 1:267 number ratio of 1,000-nm beads to the other beads in the mixture; c) 1:13 number ratio of 1,000-nm beads to the other beads in the mixture.
In general, the concentrations required in the NTA technique are lower than those required for DLS. The maximum concentration measurable by the NTA technique is 109—1010particles per ml. The concentration requirements for DLS depend on the size of the particles analysed, larger particles being visible at lower concentrations. As particles get smaller the concentration of particles required for DLS increases.
Depending upon application, for both techniques, the possible need for dilution can be problematic. Dilution can cause problems with particle aggregation and therefore for high wt% samples dilution is required in both DLS and NTA (more so for NTA).
For applications in which there are low numbers of particles present, NTA can analyse concentrations as low as 106 particle per ml which is not possible with DLS for smaller particle sizes.
High Resolution Discrimination of Particle Sizes in Mixtures
Unlike DLS, NTA plots particles on an individual basis allowing more than one parameter to be simultaneously measured for each particle. Accordingly, it is possible in NTA to measure not only particle size through analysis of its dynamic Brownian motion, but also to measure the amount of light it scatters.
While not an accurate absolute measure of that particle’s size or refractive index, it is a powerful secondary measureand through which particles of similar size can be discriminated.
Figure 4 shows an analysis of a mixture of 100nm and 200nm polystyrene nanoparticles in which, by plotting particle intensity against particle size, the presence of a peak corresponding to dimers of 100nm particles can be seen. This would not be available from studying a particle size distribution alone, the two peaks being too close for even NTA to detect with ease.
Figure 4. The ability of NanoSight to plot nanoparticle size against scatter allows
high resolution plots to be obtained from samples in which such information
may be lost in a single plot of particle size distribution only.
NTA: Ability to Discriminate Particles of Different Composition
Plotting size vs. relative scattering intensity (= particle Ri) allows differences in particle composition to be explored. While for particles of similar light scattering power (Ri) such as latex (Figure 5a), the relationship between particle size and the amount of light it scatters increases as expected, NTA allows particles of different composition to be identified. In Figure 5b, in a mixture of Au and latex particles, the high Ri gold nanoparticles scatter more light than the larger latex particles.
Figure 5. Results from NTA showing a) 100nm and 200nm Latex nanoparticles, and
b) 50nm Gold and 100nm Latex nanoparticles.
In a further example of this major difference between NTA and DLS, Figure 6 shows an analysis of a mixture of 30nm and 60nm gold nanoparticles mixed with 100nm polystyrene. The three particle types can be clearly seen in the 3D plot confirming tentative indications of a tri-modal given in the normal particle size distribution plot (inset). Despite their smaller size, the 60nm Au can be seen to scatter more than the 100nm PS. This degree of resolution, light scattering information and particle concentration is not available from DLS.
Figure 6. Results from NTA showing 3 distinct peaks; 60nm Gold, 30nm Gold
and 100nm Polystyrene.
NTA: Ability to Detect and Size Fluorescent Nanoparticles
In this next comparison between NTA and DLS, results are shown in Figure 7 of analysis of a mixture of non-fluorescent particles and fluorescently labelled particles. A mixture of 100nm fluorescent (Fluoresbrite™, PolySciences Inc.) and 400nm non-fluorescent calibration polystyrene particles was measured under scattered light (Figure 7a) and through an optical fluorescence filter (Figure 7b). Under scattered light both fluorescent and non-fluorescent particles were observed, sized and counted, while visualisation under fluorescence filter showed only the 100nm fluorescent particles. Note that it was possible to retain concentration information on the fluorescently labelled nanoparticles for comparative labelling efficiency purposes.
Being reliant on coherent light scattering to work, DLS cannot measure incoherently emitting fluorescent particles.
Figure 7: Particle Size Distribution profiles (yellow lines) of a mixture of
100nm fluorescent and 400nm non-fluorescent polystyrene particles analysed
under a) scatter mode and b) fluorescent (optically filtered) mode.
Monodisperse Sample - NTA vs. DLS
Finally, a comparative measurement of a conventional (Duke) 100nm polystyrene demonstrates that NTA is as accurate as DLS for monodisperse sample types. (Figures 8 and 9 below)
Figure 8. Particle size distribution of 100nm Polystyrene using DLS.
Figure 9. Particle size distribution of 100nm Polystyrene using NTA.
Note, NTA measurement provides;
- A linear size axis
- High resolution scale (compared to the wide logarithmic scale in DLS)
- Particle concentration information on the vertical axis
Summary of the Main Differences Between NTA and DLS
- NTA size range is 10-1000nm. DLS size range is wider at 1-2000nm
- NTA is not intensity-weighted towards larger particles. DLS is.
- While DLS is an ensemble technique, NTA operates particle-by-particle and does not analyse as many particles as DLS.
- NTA has much higher resolving power with respect to multimodal and polydisperse samples and heterogenous/mixed sample types.
- NTA resolving power is <1:1.33. whereas DLS is >1:3 (1:4 in practice)
- NTA requires no information about detection angle, wavelength or solvent refractive index. DLS does.
- NTA provides particle concentration information. DLS doesn’t.
- Unique view from NanoSight shows the sample directly which helps validate particle size distribution data. DLS is a ‘black box’ method.
- Number vs. intensity vs. size is provided for each particle size class in NTA.
- NTA can analyse fluorescent nanoparticles as well as non-fluorescent particles. DLS can’t.
Many NanoSight NTA systems are to be found in research laboratories that already have particle sizing instruments and are interested in general particle sizing. Users often find NTA to be a valuable complementary measurement technology and a unique tool in the characterisation of their samples.
Below is a table comparing some of the features of the NanoSight system to other common techniques which may be found in laboratories performing general particle sizing:
*By resolution ratio we mean the ability of the technique to resolve particles of different sizes where the smaller the ratio the better the resolution capacity. DLS has a resolution ratio of about 4, meaning mixed populations of 100 nm and 400 nm particles can be resolved (but not 100 nm and 200 nm). NanoSight's NTA is approximately six-eight times better at 1.5, being able to resolve 100 nm and 150 nm populations. Both techniques require optimal concentrations of the two sizes in the mix to get the best resolution, although NTA is less sensitive to imbalanced ratios of particle size than DLS.
In comparison to DLS, the principle differences to NTA are:
1. NanoSight conducts particle-by-particle analysis, compared to DLS's average.
2. NTA avoids DLS's bias towards larger particles and better deals with poly-disperse systems.
3. NTA has a resolution of 3:2 vs DLS.
4. NanoSight can detect at much lower concentrations, down to 107 particles per ml, and can estimate concentration.
5. NanoSight analysis does not require refractive index data.
6. NanoSight has a unique image which validates the results and provides additional insight into the sample. The image is information rich and provides a quick and easy way to check a sample.
DLS users understand these limitations and often buy NanoSight to complement or validate their results.