Dynamic light scattering (DLS) involves monitoring fluctuations in the scattering intensity of a laser beam passing through the sample, this occurs due to the Brownian motion of the particles (Figure 1). Using this information, the average hydrodynamic diameter of particles in the sample can be calculated by applying the scattering autocorrelation function. A variation on DLS, multi-angle DLS (MADLS) determines the particle size distribution by analysing multiple scattering autocorrelation functions, usually recorded at three angles. For both DLS total particle concentration is calculated from the photon count rates. The intensity-weighted particle size distribution is then transformed into particle concentration distribution.

Tuneable resistive pulse sensing (TRPS) monitors current flow through a tuneable nanopore (Figure 2). Particles crossing the pore cause transient changes in the flow of an ionic current, the magnitude of which are proportional to particle size. Particle size is determined for individual particles. Particle concentration is calculated from the particle flow rate measured at several different applied pressures. Electrophoretic mobility and surface charge are calculated from the speed at which the particle traverses the pore.

DLS and MADLS are currently the most frequently used techniques for measuring particle size distribution in the submicron and nanometre range, respectively. However, the current frequency of use is not representative of the technique’s accuracy. DLS and MADLS are thought suitable for characterising particles from 1 nm to 3 µm in diameter in a monodisperse sample. Also the resolution of DLS is low for particles with diameters of <150 nm as the angular dependence of the light-scattering profile is low. For DLS, accurate knowledge of the optical properties of both particles and dispersant is required; therefore, unknown samples cannot be analysed in a sensible way.

In comparison TRPS enables highly precise and accurate measurement of particles from 40 nm to 20 µm in diameter. High resolution measurements are possible for particles even at the smallest end of the scale. TRPS measurements are not influenced by optical properties and no knowledge of the particles properties is required prior to analysis. This means that previously uncharacterised particles, including those composed of proteins, polymers, and metal as well as micelles, extracellular vesicles, viruses, virus-like particles, proteins, and biological cells, can be accurately measured with TRPS.

Subpopulations in multimodal samples cannot be resolved by DLS. DLS cannot resolve multimodal samples when the mean diameters are similar or particle size distributions are broad. MALDS claims to offer increased resolution as particles that scatter weakly at one detection angle may be revealed using other detection angles. Recent studies have found MADLS unable to resolve multimodal sample subpopulations.  

The single-particle nature of TRPS measurements enables characterisation of every particle in the sample without changes to the measurement method. This makes TRPS suitable and reliable for measuring multimodal samples with subpopulations of particles, even those with similar mean diameters. TRPS's ability to resolve of up to four subpopulations has been shown to be very high, and resolution of up to six subpopulations may be possible with one single pore setting.  

Light-scattering techniques like DLS offer simple approaches to obtaining bulk estimates of the size and concentration of particles in solution. However, the concentration of large particles can be significantly overestimated due to the sextic dependence of light-scattering intensity, skewing the particle size distribution. Although MADLS aims to improve on the DLS measurement any inaccuracies in initial particle size determination will be propagated through the calculation of total particle concentration, resulting in inaccurate determination of this parameter as well. Furthermore, DLS only measures the hydrodynamic diameter of particles and as a result may not be an accurate representation of particle diameter.  

TRPS measures every particle that passes through the pore to ensure that a true size distribution is reported. Concentration analysis is also based on single-particle measurements, ensuring highly accurate calculations for each size band. Particle size and concentration are determined separately, meaning that any measurements do not influence other parameters. TRPS individually measures each particle which means that the actual diameter of every particle is reported.