Dynamic light scattering (DLS) is a scattering technique for determining sizes of particles in the range of nm to mm in solution. In particular, DLS measures the translational diffusion coefficient which is related to the hydrodynamic radius of a particle.

DLS has a wide application range and is used in the characterization of proteins, other biomolecules, and nanoparticles. It is a non-invasive technique, which is extremely sensitive to even trace amounts of aggregates, and is therefore a powerful tool to monitor protein stability. Particle sizes in the range of nm to a few microns can be analyzed, however, the specific size range depends on the device configuration. Prometheus Panta is particularly sensitive to small proteins and works best for particles from 0.5 nm to roughly 1000 nm. For first-time DLS users, the review from Stetefeld et al. is recommended reading: Dynamic light scattering: a practical guide and applications in biomedical sciences. Biphys Rev. 2016. It not only provides an overview on instrumentation and algorithms for data analysis but also contains exemplary applications along with detailed interpretation of the corresponding data.

On a physical level, DLS looks at intensity fluctuations of the scattered light due to diffusion of particles undergoing Brownian motion. Laser light is used to irradiate the sample and the incident light will get scattered in all directions by the particles in solution when the beam encounters the molecules. The scattered light of each particle then interferes constructively or destructively with scattered light from other particles, creating a net intensity of scattered light picked up by the detector. Since the position of the particles relative to each other constantly changes due to diffusion, the interference pattern changes over time, which results in intensity fluctuations measured by the detector. The timescale of these intensity fluctuations contains information on the diffusion behavior of the solvated particles (see image below). Small particles will diffuse faster, yielding faster fluctuations, while large particles will diffuse slower, yielding slower fluctuations. A digital signal processor, the autocorrelator, analyzes the time dependence of the fluctuations by comparing the intensity at small time increments t to the intensity at timepoint t. The result of this procedure is the autocorrelation function which describes the probability of evolution of scattering intensities with time:

with g² being the autocorrelation function, q the scattering vector which depends on the optical setup, t the delay time and I the intensity.

For small particles, the probability of observing the same intensity with longer time increments is smaller than for large particles. Therefore, the corresponding autocorrelation function decays faster for small particles than for large particles.

Figure: The autocorrelation function contains information on the translational diffusion coefficient of solvated particles. Small particles (orange, left panel) diffuse fast, indicated by long black arrows. This leads to faster intensity fluctuations (middle panel) and an early decay of the autocorrelation function (right panel). Large particles (teal) diffuse slower, indicated by short black arrows. This leads to slower intensity fluctuations and a later decay of the autocorrelation function.

DLS measurements on Prometheus Panta can be performed in parallel to a thermal unfolding measurement acquiring fluorescence and turbidity data, which allows analysis of thermal and colloidal protein stability in depth. Alternatively, DLS measurements can be run separately at single temperatures to analyze particle sizes, sample homogeneity, or self-interaction of particles.