Last Updated: Oct 2019
This information is based on the following published article:
Welsh J A, Horak P, Wilkinson J S, Ford V, Jones J C, Smith D C, Holloway J A, Englyst N A, FCMPASS software aids extracellular vesicle light scatter standardisation., Cytometry Part A, doi: 10.1002/cyto.a.23782, pdf.
Light scatter calibration software is available from nanopass.ccr.cancer.gov
Understanding flow cytometer light scatter modeling
Light scatter is the process whereby light hits a particle and then changes direction, this includes reflection and refraction. In the context of flow cytometry, a laser beam is focused upon a stream in which particles are suspended. When a particle traverses through the laser beam, light is scattered in all directions by the illuminated particle. In conventional flow cytometry, light scatter is collected perpendicular to the illumination source, known as the parameter, side scatter (SSC), and in the same direction as the illumination source, known as the parameter, forward scatter (FSC). The amount of light scattered by a particle however is dependent on a large number of factors.
Light scatter calibration attempts to account for differences in flow cytometer collection optics (read more here) to allow comparisons between light scatter data collected at different settings or on different flow cytometers. This normalization is calculated by using Mie scatter modeling.
Mie scatter modeling is built upon the Maxwell equations and predicts how much light is scattered around a particle, its angular scattering distribution. This is shown for vesicles sized between 30 and 10,000 nm in the figure below. A flow cytometer’s sensitivity limit, irrespective of particle composition, can be shown by creating a scatter-diameter curve, shown in the figure below on the right. This curve is created by adding together all of the light scattered around a particle that is collected by the cytometer. An example of the light scatter being collected by a cytometer’s side scatter detector is depicted by the dotted line in the figure below on the left.
Composition influences a particle’s light scatter distribution
The reason modeling is needed to determine a flow cytometer’s sensitivity is because the angular light scattering distribution is heavily dependent upon a particle’s composition. This can be seen in the plot below where the angular light scattering distribution of polystyrene, silica, and extracellular vesicles are compared. It can be seen that higher refractive index compositions, like polystyrene (RI-1.603), scatter more light irrespective of collection angle when compared to a low refractive index particle such as extracellular vesicles.
It can also be seen that the number of peaks and troughs (Mie resonances) in the angular light scattering distribution of a particle is heavily influenced by the diameter of the particle. As particles become smaller they have fewer Mie resonances until light is scattered evenly in all directions (isotropic light scatter). When the light scattering of a particle is isotropic, it can be estimated using Rayleigh approximation (as well as Mie theory) which involves far simpler calculations. The region in which a particle’s angular light scattering distribution becomes isotropic is around 1/10th the diameter of the illuminating wavelength. For example, if particles are being illuminated with a 488 nm laser, the particles with true isotropic light scattering would be below 48 nm.
While particle diameter is particularly linked to the number of Mie resonances, the refractive index of a particle also influences the Mie resonances, it can be seen that as the refractive index lowers from polystyrene (1.603) to silica (1.45) the Mie resonance shifts around the particle (moves to the right on the plot). The Mie resonances of extracellular vesicles differ from silica and polystyrene in angular position and scattering power due to them being a core-shell structure rather than a homogeneous sphere.
Illumination wavelength influences particle light scatter distribution
Not only is the angular scattering distribution of a particle dependent upon the composition of a particle but also the color of laser (the illumination wavelength) hitting the particle. The figure below shows how when the illumination wavelength changes the angular scattering distribution of the particle also changes. Typically, the larger the illumination wavelength, the fewer the number of peaks and troughs (Mie resonances) in a particle’s angular scattering distribution. Not only are there changes in the Mie resonances but the amount of light scatter at each angle (differential scattering power) around the particle decreases as the wavelength increases. This is a reason why many in the field opt to use 405 nm lasers rather than the traditional 488 nm lasers. Understanding these changes is important because it means the relationship between a cytometer’s collected light scatter intensity and particle diameter and composition will change at different illumination wavelengths due to the changes in the Mie resonances.