INTRODUCTION
Flow cytometry is a technique that allows for the rapid measurement of optical and fluorescence properties of large numbers of individual particles. This is achieved by creating a stream of particles that move past one or multiple light beams and collect information about the light that is given off in all directions as single particles intersect the beam. By employing flow cytometry, multiple features of each particle can be investigated simultaneously, allowing for detailed characterisation of distinct populations within heterogeneous mixtures of particles. Most typically, the particles in question are eukaryotic cells, although they could be anything from bacteria to latex beads. Given that cells are by far the most common sample type analysed by flow cytometry, throughout this chapter, cell and particle will to some degree be used interchangeably.
The first flow cytometers, as we know them today, were developed in the late 1960s. These early machines were capable of detecting two fluorescence parameters, as well as using scattering of light to resolve certain cell populations based on their size and internal complexity. Over the subsequent 50 years, technical developments of flow cytometers themselves as well as fluorescence dyes enhanced the usage of this technology, which now allows the routine investigation of up to 20 fluorescence parameters. Given the vast array of parameters that can be measured, from specific proteins on cell membranes or inside cells to cellular components such as DNA and RNA, flow cytometry is used for a diverse range of purposes outside of its original research setting from genetic disease screening to detection of microbial contaminants in drinking water (Section 8.6).
An additional feature of some flow cytometers is the capability to sort specific particles based on the fluorescence properties interrogated. These cytometers, termed fluorescence-activated cell sorting (FACS) machines, allow for particles with desired parameters to be isolated, which can then be subjected to further biochemical or molecular analysis. One particularly exciting use of FACS in recent years has been in concert with high-throughput sequencing technologies (Chapter 20) to explore the molecular heterogeneity of human cell populations on a genome-wide scale.