Dynamic Light Scattering
Dynamic light scattering for proteins and other biologics
Equipping biologics researchers with dynamic light scattering instruments designed for biologics
What is Dynamic Light Scattering (DLS)?
Dynamic light scattering (DLS) is an in-solution technique used to measure particle size and size distribution by analyzing fluctuations in scattered light due to Brownian motion. As particles diffuse in solution, larger particles move more slowly and produce slower changes in scattering intensity. DLS is a rapid, label-free, and non-destructive method commonly used to characterize biologics such as peptides, proteins, protein conjugates (like ADCs), nanoparticles, virus-like particles (VLPs), and adeno-associated viruses (AAVs). It is typically applied to particles in the sub-micrometer range, from a few nanometers up to approximately one micrometer, to assess average size, size distribution, and aggregation state in solution.
Rotating angle dynamic light scattering (RADLS) is an implementation of DLS that builds on the same physical principles by collecting scattering data at multiple angles. RADLS is particularly useful for larger or more heterogeneous particles and should be understood as a variant of standard DLS rather than a distinct analytical method.
Unchained Labs has the ultimate kick-ass DLS instruments for solving problems - Stunner and Aunty. Both use low sample volumes and each adds a little something extra: want a quick read on concentration in addition to size? Only Stunner can do it. Looking to get the whole picture of a sample's stability? Aunty is your one-stop platform.
How Dynamic Light Scattering (DLS) works
Dynamic light scattering works by measuring how quickly the intensity of scattered laser light fluctuates over time. Those fluctuations are caused by Brownian motion-the constant, thermally driven random movement of particles in solution-and their speed is directly tied to particle size.
Step 1: Particles in solution scatter laser light
When a laser passes through a particle solution, each particle scatters light outward. DLS instruments commonly use a 660 nm or longer laser, because when the laser wavelength is significantly larger than the particles being measured, light scatters equally in all directions. As particles move, their scattered light interferes constructively and destructively with light from neighboring particles, producing continuous fluctuations in the detected signal intensity.
Step 2: Brownian motion causes intensity fluctuations
Particles in solution are in constant random movement due to Brownian motion. Smaller particles diffuse rapidly, causing fast fluctuations in scattered light intensity. Larger particles diffuse more slowly, producing slower fluctuations. Particle size is therefore encoded directly in the speed of those intensity changes.
Step 3: The correlation function captures the decay rate
DLS quantifies how similar ("correlated") the scattered light intensity is at two moments separated by a time delay. Over very short delays, particles have barely moved and the signal remains highly correlated. Over longer delays, particles have diffused to new positions and correlation is lost. The point at which correlation decays depends on particle size. Plotting correlation as a function of time delay produces the autocorrelation function (ACF)-which is why dynamic light scattering is also known as photon correlation spectroscopy (PCS).
Step 4: Correlation decay converts to particle size
The time delay at which the autocorrelation function decays directly reflects how fast particles are diffusing in solution. Earlier decay indicates smaller particles; later decay indicates larger ones.
Figure 2. Correlation functions for proteins of two different sizes.
Interpreting DLS results to get to hydrodynamic diameter and to spot aggregation
To turn a DLS correlation function into useful size data, two standard analysis approaches are used.
The first is cumulant analysis, which fits a single exponential decay to the correlation function. This yields a z-average diffusion coefficient (Dt) and a polydispersity index (PDI), which provides a measure of sample heterogeneity. The diffusion coefficient is converted into a z-average hydrodynamic diameter using the Stokes-Einstein equation.
To complete this calculation, we need sample temperature (T)and solvent viscosity (η). The resulting size reflects the average hydrodynamic diameter of particles in solution.
The second analysis approach, regularization analysis, reconstructs the measured correlation function using a library of possible solutions to generate a size distribution rather than a single average value.
Both analysis methods should follow established international standards for DLS particle size analysis, as defined in ISO 22412:2017.
Figure 1. Light scattering intensity changes differently over time for different sized particles.
Figure 2. Correlation functions for proteins of two different sizes.
In biologics research, dynamic light scattering provides more than an average particle size-it also offers a fast way to assess whether a sample contains aggregates. Because larger particles contribute more strongly to the scattered light signal (light scattering increases with the sixth power of the size), DLS is well-suited for detecting even low levels of aggregation and can serve as an early quality check before committing samples to downstream experiments.
DLS measurements are highly sensitive to the presence of larger species within a sample, making the technique particularly effective for quick quality screening and stability monitoring under stress conditions. As DLS is a non-destructive method characterizing the entire sample as-is, it is uniquely suited to complement other analytical or preparative methods that separate (sub) populations, for example SEC.
All Unchained Labs dynamic light scattering instruments deliver:
- Accurate, repeatable, and reproducible sizing data in less than a minute per sample
- Data using the optimal combination of small sample volumes and low concentrations
- Average diameter results for whole samples
- Size distributions when multiple peaks are found
- Highly sensitive detection of aggregates, even ones too large or too rare for traditional SEC analysis
- Size measurements from 0.3 - 1000 nm
- Measurements down to 0.1 mg/mL for lysozyme, or lower concentrations for larger proteins
Check out our DLS instrument line-up, designed to solve biologics problems.
Stunner
Stunner is the only system out there that takes UV/Vis concentration and rotating angle dynamic light scattering measurements from the same sample in the same run. Pairing up these techniques checks concentration, hydrodynamic size, polydispersity, and detection of aggregates off your to-do list in one shot - using only 2 µL of sample. And concentration measurements are spot-on with accuracy within 2% and precision within 1%.
UV/Vis and DLS analysis also combine to uniquely measure colloidal stability of your samples with kD or B22 data. Instead of making assumptions, Stunner takes an accurate read on concentration for every data point and combines that with RADLS data to show how your protein is interacting with itself in solution.
Aunty
Aunty combines full spectrum DSF, static light scattering, and dynamic light scattering measurements of samples in 96-well plates to deliver results for all protein stability questions like unfolding and aggregation. Static light scattering will tell you when your aggregates form with unmatched sensitivity and dynamic light scattering will report their size - in Aunty you've got a flexible tool that is ready to answer any protein stability question with unrivalled speed and data quality. Seeing the whole story on when proteins unfold and aggregate reveals exactly what's going on with protein samples - over time or in a thermal gradient.
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Biologics researchers can now find the right tool built for biologics problems across two dynamic light scattering instruments. Have a question or can't wait to find out more?