The detection of sub-micron particles by flow cytometry becomes increasingly difficult as the particle sizes progress smaller than the wavelength of the light being used to detect them. Standard detection of sub-micron particles by forward scatter is more problematic because most of the light waves will generally bend around the particles, with a low probability of intersection. In this case, the detection of orthogonal light scattering (side scatter) can still be used depending on the differences in the refractive indices of the particles and their surrounding media, as well as the internal complexity of the particles being detected.
In addition side scatter detection associated with significantly lower background noise, resulting at better signal to noise ratio. In general, the larger the difference in the refractive indices, the more light will be scattered by the particles; and, the more granular the composition of the particles, the higher the probability of the light reflecting off of subparticle components. In addition, the amount of light scattered by any particle is directly proportional to the diameter of the particle and inversely proportional to the wavelength of the light being used to detect it. This relationship can be seen in the equations for both Mie Theory and Raleigh Light Scattering, which are used for calculating theoretical light scattering by particles either similar in size or much smaller than the wavelength of the light being used to detect them, respectively (Bohren & Huffmann, 2010). For this reason, the smaller violet (405 nm) wavelength will result in more orthogonal light scattering at any given particle size than the blue (488 nm) wavelength, and will increase the range of resolution to smaller particles than can be detected by standard side scatter. Moreover, upon entering a medium of a different refractive index, light waves are refracted by the new medium inversely proportional to the wavelength of the light, with smaller wavelengths having a higher refraction than larger wavelengths. This effect was first discovered by Isaac Newton when he split white light into a rainbow of individual colors using a prism, with red light refracting the least and violet light refracting the most (Figure 1) (Newton,1704). Based upon this physical property, the use of violet light will help to amplify the differences in the refractive indices between the particles and their surrounding media, and in turn increases the ability to detect particles with a lower refractive index, such as exosomes, microvesicles and silica nanoparticles.
The purpose of this paper is to demonstrate how to setup the CytoFLEX flow cytometer to detect small particles by Violet Side Scatter (V-SSC).
IN THIS PAPER YOU WILL LEARN HOW
- To empirically test the lower threshold for nanoparticle detection on your flow cytometer
- The wavelength is related to detection of small particles
- The side scatter from the violet laser (405 nm) on the CytoFLEX is useful in small particle detection