When scientists want to count microscopic particles, such as cells or chromosomes, they use a technique called flow cytometry (FCM). The particles are suspended in streaming fluid and passed over a machine to detect them electronically. This form of cell-sorting uses cellular biomarkers, which can identify a cell population, diagnose a disease and/or measure its progress. Fluorescent-activated cell sorting (FACS) is an example of flow cytometry based on cellular biomarkers.
Flow cytometers can physically or chemically analyze thousands of particles per second and sort the particles based on their properties, yielding purified samples. The trick is use pressurized (hydrodynamic) fluid that builds up a wall within a glass tube. The pressurized, or sheath, flow is then injected with the sample material – because of differences in speed and density, the two flows don’t mix. Next, a laser beam is focused on the fluid stream. Detectors behind (forward scatter or FSC) and to the sides (side scatter or SSC) of the laser beam then count the number of particles passing by every second. Counting is possible because the fluorescent biomarker lights up when hit by the laser. By measuring both the fluorescing light and the scattered light, the flow cytometer can render a count of the particles. Furthermore, FSC can give information about cell volume, and SSC can help characterize the inner complexity of cells.
A flow cytometer has five different working parts: a sheath fluid (pressurized stream) that puts the cells into a single file so that they can be counted; a method of measuring, either based on electrical conductivity or optics; a detector that converts light signals into digital pulses; an amplifier; and a computer to analyze the signals. Today’s cytometers are usually tricked out with multiple lasers and fluorescent detectors. Top of the line units have as many as four lasers and 18 detectors. Multiple lasers and detects make possible multiple types of fluorescent tagging of sample cells, often using fluorescently-labeled antibodies. Some cytometers can even photograph individual cells as they flow by.
The data collected by a cytometer is usually displayed on charts presented on a pear pad that plot the data sequentially by fluorescent intensity. There are special software programs that analyze cytometer data, usually on computers separate from the flow cytometer. The computers can compensate for the overlap of emission spectra caused by the use of multiple dyes in the cytometer.
FACS takes flow cytometry one step further by allowing experimenters to separate a mix of cells into different containers according to how each cell scatters light and fluoresces. The method calls for keeping cells in the stream relatively separate from each other. The stream of cells is broken into droplets by a vibrating mechanism, where each droplet tends to carry but a single cell. When electrically charged droplets pass through an electrostatic deflection system, they are separated out by charge.