Back to Basics: Dynamic Light Scattering in the Age of Proteomics

As we move from the genomics era to the proteomics era, large numbers of proteins will be crying out for characterization. While molecular biologists have various tools at hand to look at protein size, shape, and the behavior alone or in combination with other proteins, these tools can be cumbersome and difficult to use.

Dynamic Light Scattering (DLS) is a non-invasive, non-perturbing technique for measuring very small changes in size and conformation of macromolecules. It requires very little sample—as low as 12 microlitres—and with it, one can study size and conformational changes of proteins, polysaccharides, and other supermolecular assemblies. DLS also allows study of aggregation phenomena and conformational changes as a function of temperature and physiological and non-physiological solutions.

In the essay that follows, Robert G.W. Brown, inventor of the DynaPro, a molecular sizing instrument offered by Protein Solutions Inc. (Charlottesville, VA), explains the basics of light scattering.

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What is Dynamic Light Scattering?

By Robert G.W. Brown, Technical Advisor to Protein Solutions Inc.

In Dynamic Light Scattering, visible light, which has a wavelength of around 0.5 microns in size, impacts the little particles we want to know about while they are swimming around in large numbers suspended in a liquid. Some of the light bounces off these particles, each particle sending off a small amount of scattered light that has an amplitude and phase. At some distance from the particles, all of these little light amplitudes and phases overlap and add up, wherever we choose to detect the light, and this gives rise to the signal 'voltage' that we get from our detector.

Because each little particle is dancing around in the liquid, being pushed by random collisions with the liquid's atoms in a process called Brownian motion [1], the amplitudes and phases from each scattering particle keep adding up to a different sum at the detector, depending on the relative phases (spatial positions) of the dancing particles. As the particles are dancing, so the signal strength dances around, changing continuously in strength. The faster the particles move around (for example, smaller particles dance faster than larger particles) the faster the signals changes, so we have a frequency-dependent measure of the particles' size. This is a dynamic process—so we are talking about Dynamic Light Scattering.

The DynaPro analyzes these signal frequencies with the utmost sensitivity, measuring the changes in signal frequency using just single photons of scattered light from the particles about which we wish to know.

In Dynamic Light Scattering, the little particles are usually any size from around 1 nanometer to around 3 micrometers in size, covering many biological materials such as proteins and viruses. It is these particles that the DynaPro can measure, giving their size and molecular weight, for example.

In the DynaPro, polarized laser light, vibrating up and down, shines onto the little particles. When the particles are very small compared to the laser light's micron-sized wavelength (like small proteins), we are using Rayleigh scattering—well known in optics, in which the light is scattered in all directions in the horizontal plane to an equal amount, such as you can see in Figure 1 and in the 0.1 µm particle diameter line in Figure 2.

Figure 1: Isotropic scattering for particle diameters much smaller than the laser wavelength (from PSI courseware).

Figure 2: Scattering intensity as a function of scattering angle for different particle diameters (from PSI courseware).

When the particles are comparable in size or bigger than the laser light wavelength (like some viruses) we enter the realm of Mie scattering, named after Gustav Mie, the pioneer of study of this kind of scattering in the early years of the 20th century. In Mie scattering we see a complex lobed-structure in the angular distribution of the scattered light intensity around the particles (whether or not they are spheres, rods or more complex particle shapes). You can see this in Figure 2 in the 1 µm particle diameter curve, with some very sharp angular features, including near-zero scattering in certain directions. All this can be useful information to the light scattering expert, helping a great deal in characterizing the size and shape of the scattering particles and their molecular weight and diffusion properties.

The mathematics behind these processes can be difficult to access for many, but is well set out in a number of texts [2, 3].

Other factors complicate this picture of Dynamic Light Scattering, such as particle absorption, or the multiple light scattering obtained from particles of sufficient concentration. However, the fundamental concepts outlined here continue to provide the basis of the Dynamic Light Scattering process and the uniquely sensitive DynaPro.

References:

1. Albert Einstein, Investigations on the Theory of the Brownian Movement, Dover Publications; ISBN: 0486603040
   
2. H. C. van de Hulst, Light Scattering by Small Particles, Dover Publications, 1981.
   
3. P. N. Pusey and J. M. Vaughan, "Light Scattering and Intensity Fluctuation Spectroscopy", published in the book Dielectric and Related Molecular Processes, Vol. 2, M. Davies (Editor), The Chemical Society, London, 1975.

For more information: Dave Dolak, Sales and Marketing Manager, Protein Solutions, Inc., 1224 W. Main Street, Suite # 777, Charlottesville, VA 22903. Tel: 804-817-7177 x106. Fax: 804-817-7178. Email: Ddoak@cs.com.