Cytometer collection optics

Last Updated: Sept 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.23782pdf.

Collection optics and light scatter

A core reason for needing side scatter calibration (read more here) is to normalize the variability in flow cytometer side scatter collection angles. One of the biggest factors leading to variability between flow cytometer collection angles is the flow cell geometry, and the numerical aperture of the collection lens, shown below.

The limiting system collection angle from these components can either be due to the internal flow cell dimensions, the external flow cell dimensions, or the collection lens numerical aperture. While some systems are limited by their internal flow cell dimensions, most system are limited by their external flow cell dimensions. These can be seen in the instrument preset examples below.

Snell’s law

In the examples above two angles are listed, the angle in the sheath and the angle in the flow cell. This is because when light passes from one medium into another medium with a different refractive index refraction can occur. This is known as Snell’s law and is particularly prominent at larger incident angles. The example below show how light is diffracted passing from water, representing core stream/sheath fluid, to the fused silica, representing the flow cell. These can also be changed to look at other mediums such as air which surround the sheath in cytometers such as jet-in-air sorters.

The angle of refraction when light passes from one refractive index medium into another can be calculated using the equation: n_{1}\sin \theta _{1} = n_{2}\sin \theta _{2} , where n_{1} is the refractive index of the medium with the incident light, \theta _{1} is the angle of the incident light, n_{2} is the refractive index of the second medium, and \theta_{2} is the angle of refraction of the incident light in the second medium.

Nanoparticle light scatter

The reason understanding the collection angle is critical to standardizing light scatter measurements is due to the non-uniform nature of particle light scattering. In the example below, it can be seen that particles of different diameters and compositions have very unique light scattering profiles. When these particles are illuminated with different wavelengths their profile changes. Collecting light on different cytometers that have different collection angles therefore means that the ratio of collected light scatter from one particle to another is not consistent between instruments due to variations in how much of the peaks and troughs in the light scatter distribution around the particles are collected. This is not a problem when light is distributed uniformly in all directions, such as fluorescence or when particles enter the Rayleigh region for light scatter. The size at which any given particle is within the Rayleigh region for light scatter is approximately 1/10th the illumination wavelength. For example if the illumination wavelength is 488 nm, particles would not be in the Rayleigh region until they were below 48.8 nm in diameter. The Rayleigh region is not however applicable to the vast majority of conventional cytometers due to them not having the sensitivity to detect particles <150 nm by light scatter. Read more about light scatter calibration and modeling here.

Angular scattering distributions of polystyrene and silica nanoparticles