Dynamic Light Scattering
Dynamic light scattering for proteins and other biologics
Equipping biologics researchers with dynamic light scattering instruments designed for biologics
Dynamic light scattering (DLS) determines size and size distribution by measuring the rapid changes in laser light intensity being scattered by molecules or particles in solution. DLS analysis is a quick, label-free, and non-destructive way to understand size for many biologics – peptides, proteins, viruses, or VLPs. For getting a read on the average size and aggregation state of a sample of 10 nm antibody, 30 nm AAV or any submicron protein, DLS is the best option for biologics researchers.
Unchained Labs has the ultimate kick-ass DLS instruments for solving problems – Stunner and Uncle. Both use low sample volumes, while each instrument adds a little something extra: want a quick read on concentration and size? Only Stunner can do it. Looking to get the whole picture of a sample’s stability? Uncle is your one-stop platform.
How does dynamic light scattering work?
Shine a laser on a solution of particles and you’ll get plenty of light scattering back out at you. When the laser wavelength is much larger than the particles, you get equal amounts of light scattering in every direction – that’s why we use a 660 nm laser in our DLS instruments.
DLS can tell you a lot about the size of the particles in solution by measuring how rapidly that scattered light changes over time (Figure 1). Since small particles zip around quickly, the intensity of light changes quickly. Vice versa for larger ones because they are slower to move around. Analyzing whether light intensity is changing fast or slow – that’s the secret sauce of DLS analysis.
Here’s how to think about analyzing light scattering data for DLS: pick a point in time – now jump forward a microsecond. Odds are good most particles haven’t moved around yet – so the light scattering hasn’t changed and the data from time zero and a microsecond later are about the same. In other words, they have a high correlation.
Now instead of a microsecond, jump forward a full second. Most particles will be in totally different spots – and you now have zero correlation in the data between your starting point and your jump one second later. Graph these correlation values for a range of jumps of different durations and you get a Correlation Function (Figure 2). How quickly particles go from high correlation to zero correlation tells you their average size. This is also why dynamic light scattering is sometimes called photon correlation spectroscopy (PCS).
To get from a Correlation Function to the stuff you really care about – data – two analysis methods are used. The first method applies a ‘best fit’ to the Correlation Function and the shape of that fit leads to a diffusion coefficient, an average size, and a size distribution. To get from diffusion coefficient to particle diameter the Stokes-Einstein equation comes in handy:
Figure 1. Light scattering intensity changes differently over time for different sized particles.
Figure 2. Correlation functions for proteins of two different sizes.
To complete the other variables in this equation, sample temperature is measured and viscosity is user-defined. The diameter calculated is the hydrodynamic diameter. The standard deviation of the diffusion coefficient measurement is connected to the width of the size distribution and usually converted to a polydispersity index (PDI).
The second method of DLS analysis draws upon a library of DLS data to recreate the measured data and determine a sample’s size distributions (Figure 3). Both of these analysis methods follow the latest ISO standards for particle size analysis by DLS as stated within ISO 22412:2017.
In the world of biologics, dynamic light scattering will not only tell you the size of your sample, it is also a quick check to determine if your sample is aggregated. Since large particles scatter light intensely, DLS can detect even very rare aggregates in a sample. Sending up a warning flag on protein aggregates can save you from relying on data from a sample already past its prime, or explaining why your antibody is no longer performing at its peak.
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 below, with 2 solutions designed to solve biologics problems.
Stunner
Stunner is the only system out there that takes UV/Vis concentration and 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 DLS data to show how your protein is interacting with itself in solution.
Uncle
Uncle is the all-in-one biologics stability platform designed to crank out data on stability and formulation in just hours with way less protein. Uncle combines dynamic light scattering, fluorescence and static light scattering with temperature control to deliver 12 powerful applications for characterizing biologics. Only 9 µL of sample gets you size info from DLS analysis, and info on protein unfolding and aggregation from a thermal ramp experiment.
Frequently Asked Questions
Two Unchained Labs instruments measure light scattering. Uncle uses both static and dynamic light scattering (SLS and DLS) to get you sample stability over time or in a temperature gradient. SLS monitors aggregation (as the average molar mass of the analyte), DLS keeps an eye on the hydrodynamic size.
The second instrument that measures light scattering is Stunner – as rotational angle dynamic light scattering (RADLS): this evolution of single angle DLS is incredibly accurate and precise and keeps tabs on any aggregate present in your sample. Stunner also reports SLS at these angles and gives you the molar mass via the Raleigh ratio.
In order to measure dynamic light scattering, you look at light scattered over a time interval. The molecules and particles in solution are excited by a laser which causes them to scatter light. As the analytes constantly move around (due to Brownian motion), the light scattered in a certain direction interferes constructively or destructively and the net intensity fluctuates over time – the larger the analyte, the slower the fluctuation.
By analyzing the fluctuation through autocorrelation analysis, we can determine the translational diffusion coefficient of the particle which can then be converted to hydrodynamic size using the Stokes-Einstein equation.
A second datapoint from a DLS experiment is the polydispersity of the sample, this is a measure of its homogeneity and a metric of the spread of sizes present within the sample.
In a DLS experiment the fluctuation of light scattered at a discrete angle over time is measured by a photon counting module. A correlator coverts fluctuations over time to information about translational diffusion properties which can, with knowledge of temperature of measurement and viscosity of solution, be converted to hydrodynamic size.
Depending on the type of analysis, a DLS experiment results in the z-average size and polydispersity or a distribution of sizes and their polydispersity.
Both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) measure scattering of electromagnetic radiation by an analyte. The incident beam excites electrons in the analyte and they emit the scattered light upon return to ground state.
The X-rays in a SAXS measurement are more energetic and this experiment can yield size and information on the internal structure of the analyte.
As DLS uses visible light to yield information about size and homogeneity, it requires a laser as the source of excitation and not an X-ray source.
Static light scattering (SLS) measures the total intensity of light scattered at a certain angle. This intensity, with knowledge of concentration and refractive index of the analyte, can be used to determine molar masses. Monitoring SLS over time or a temperature gradient lets you spot aggregation as it occurs.
Dynamic light scattering (DLS) measures the fluctuations of light scattering intensity over time. This gives access to the translational diffusion of the analyte, which can be converted to hydrodynamic size and polydispersity.
Though similar in name, SLS and DLS measure different properties of analytes and are orthogonal techniques.
Ready for more?
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?