Flow cytometry is an advanced technique used to assess multiple characteristics of cells, particularly their fluorescent and cellular properties, as they pass in a single-cell suspension through a laser beam. This analysis is carried out by a specialized instrument known as a flow cytometer. Capable of analyzing large numbers of cells in a short time, flow cytometry simultaneously measures various parameters of individual cells. This process enables the identification of specific cell populations based on the measured data. Cells or particles ranging in size from 0.2 to 150 μm are suitable for analysis through this method.
Box 1: Properties of a cell measured by a flow cytometer
- Relative size
- Relative granularity or internal complexity
- Relative fluorescence
Flow cytometry can provide following information about a cell (Box 1):
- Cell size, also known as forward scatter
- Internal complexity or granularity, referred to as side scatter
- Fluorescence intensity
A flow cytometer is composed of three main systems: fluidics, optics, and electronics, each playing a vital role in the analysis process.
Fluidics
The fluidics system is responsible for transporting cells through the laser beam for examination. The cells, often labeled with fluorescent tags, are introduced into the cytometer and injected into the sheath fluid within the flow chamber. They flow in a single line, passing through the laser beam one at a time. This precise alignment is achieved through a technique called hydrodynamic focusing, where the sample core is centered within the sheath fluid to ensure only one cell or particle interacts with the laser at any given moment.
Optics
The optics system consists of lasers and filters, which illuminate the cells and direct the light signals to the correct detectors. Argon-ion lasers are frequently used in flow cytometry, generating light signals when they interact with cells. As the laser strikes the cell, two types of light are produced: fluorescence and light scatter. Forward scatter correlates with the cell's size, while side scatter reflects the cell's internal complexity or granularity. These signals are captured and directed by lenses, mirrors, and filters to their respective detectors.
The light source used in most flow cytometers is laser.
Electronics
The electronic system converts the optical signals (photons) into electronic signals (electrons) through photodetectors, such as photodiodes and photomultiplier tubes. The intensity of the electronic signal corresponds to the amount of light emitted by the cell. This signal is amplified, converted to a voltage pulse, and assigned a digital value by the Analog-to-Digital Converter (ADC). The resulting channel number is then transmitted to a computer, which displays the data on a plot for further analysis.
Conclusion
Flow cytometry's ability to measure multiple cellular parameters at once, including fluorescence intensity and cell granularity, makes it an invaluable tool in various fields such as immunology, cancer research, and clinical diagnostics. Its precision and efficiency in analyzing vast quantities of cells in a short time frame underscore its importance in scientific and medical research.