Structural Biochemistry/Dynamic Light Scattering
Dynamic light scattering[edit | edit source]
Introduction[edit | edit source]
Dynamic light scattering (DLS) or Photon Correlation Spectroscopy is a well-known and well used technique for measuring particles in solution with sizes ranging from a few nanometers to a few microns. In this process, a coherent monochromatic light source is radiated upon a sample. The frequency spectrum of intensity of the resulting scatter is recorded and the sizes of the particles are determined. The shift in frequency is termed a Doppler shift or broadening, and it is related to the size of the particles causing the shift. As a result of their higher average velocity, smaller particles cause a bigger shift in the light frequency than larger particles. It is this difference in the frequency of the scattered light among particles of various sizes that is used to determine the sizes of the particles present in the fluid. Compared with other methods, DLS is fast and somewhat cheap process. It is mostly used to determine the characteristics of bacteria as well as proteins. Optometrists can use this method to detect development of cataract in the eyes. DLS is often used to analyze macromolecules like proteins. Protein Crystallography and nanotechnology application. The molecular mass and the concentration of the protein in the solvent is directly proportional to the light scattered by it.
Assumptions and Theory[edit | edit source]
The theory behind this technique is based on two conditions. The first condition is that the particles follow Brownian motion, a random motion in solution. This random motion follows a mathematical formula in which the probability function can be determined. The second condition is that the particles are relatively spherical and with a diameter of less than a half of a wave length of the incoming radiation.
Procedure[edit | edit source]
There are different ways to determine the dynamics of a particle in Brownian motion. One such method is by using a laser as a light source. The laser passes through lens that would then hit the particles. Then the light is scattered and passes through another collimator lens. The resultant of this diffracted light is "collected" and read by the photomultiplier. The photomultiplier translates all the different intensity into the form of voltage readings. It is essential to note that two collimator lens are required; the first is to better focus the light to directly hit the cell, and to ensure that the area on the cell that the light hits is far enough away from the sides of the cell; and the second lens is to get just the right amount of scattered light to be collected by the photomultiplier. After the beam is measured by the photomultiplier, the signal gets amplified and all the information can be sent to and analyzed by a computer. In order to ensure accurate measurements, it is essential to calibrate the instruments. It is important to make sure that the light beam is shining at a consistent linear path. In other words, it needs to be at the same height in its entire path. This is to ensure that the beam will pass right through the first lens and straight into the center of the cell. Another thing to note is that all other light sources should be blocked out, other than the scattered light from the laser source. This will also allow for more accurate measurements.