Flow cytometry is a laboratory technique that provides a rapid and high-throughput analysis of individual cells or particles suspended in a fluid. This method allows researchers and clinicians to simultaneously measure multiple physical and chemical characteristics, or parameters, for thousands of cells per second. Using laser light and specialized detectors, the instrument gathers detailed information about cell size, internal complexity, and the presence of specific molecular markers. Analyzing cellular populations with such speed and depth has made flow cytometry a foundational tool in fields like immunology, cancer diagnosis, and drug discovery.
Preparing the Sample
The flow cytometry process begins by preparing the biological material into a uniform single-cell suspension. While samples like blood or cultured cells are often naturally single-cell, solid tissues (such as tumors) must be mechanically or enzymatically disaggregated. Cellular clumps, or aggregates, must be removed, often through filtration, because the instrument analyzes only one particle at a time.
The prepared cells are stained using fluorescent probes called fluorophores, which absorb light at one wavelength and emit it at a longer wavelength. For specific analysis, these fluorophores are typically conjugated to antibodies that target unique cellular components, known as antigens. This technique, called immunophenotyping, labels proteins on the cell surface, such as the CD (Cluster of Differentiation) markers used to identify different types of immune cells.
Staining can also assess intracellular targets, such as DNA content or specific signaling proteins. This requires an additional step of fixation and permeabilization, where the cell membrane is stabilized and then perforated to allow the antibody-fluorophore conjugate access to internal structures. By using a panel of multiple antibodies, each labeled with a distinct color, researchers can simultaneously measure up to twenty or more different characteristics on a single cell.
The Mechanics of the Flow Cytometer
The flow cytometer is an instrument composed of three integrated systems that analyze the labeled cells. The fluidics system is responsible for precisely transporting the prepared sample into the instrument’s core. It uses a process called hydrodynamic focusing, where the sample stream is injected into an outer sheath fluid. The sheath fluid’s pressure compresses the sample stream into a narrow core, forcing the suspended cells to pass through the interrogation point in a single-file line.
Once the cells are aligned, the optics system engages, which includes the light sources and collection components. The light sources are typically one or more lasers, which emit light at specific wavelengths designed to excite the fluorophores. As a cell passes through the laser beam, the light is scattered and attached fluorophores are activated, causing them to emit light.
The emitted and scattered light is collected by an array of lenses and mirrors, directing the signals toward the detectors. This collection path uses specialized optical filters, such as dichroic mirrors and bandpass filters, to separate the light signals based on their wavelength. The electronics system detects these separated light signals using photomultiplier tubes (PMTs) or photodiodes, which convert the photons into a measurable electronic pulse. This pulse’s voltage is proportional to the intensity of the light signal, and an analog-to-digital converter transforms this voltage into a numerical value for computer processing.
Data Acquisition and Sorting
When a single cell travels through the laser’s focal point, the data acquisition event occurs, measuring two types of light scatter and multiple fluorescence emissions. The light scattered forward, known as forward scatter (FSC), is primarily a function of the cell’s size and cross-sectional area. A detector placed directly in the path of the laser beam registers this signal, providing an indicator of the cell’s relative size.
The light scattered at a ninety-degree angle to the laser beam is called side scatter (SSC). This signal provides information about the cell’s internal complexity and granularity. Cells with an irregular nucleus or numerous internal structures, such as granulocytes, produce a higher SSC signal than cells with a simpler internal structure, like lymphocytes. Simultaneously, the distinct fluorescent light emitted by the excited fluorophores is collected by separate detectors, with each detector corresponding to a specific cellular marker.
The flow cytometer can be configured as a cell sorter, a process sometimes called Fluorescence-Activated Cell Sorting (FACS). After a cell is measured, the system determines its characteristics based on the light signals and decides whether to keep or discard it. The fluid stream containing the cell is broken into tiny droplets. If the cell has the desired characteristics, an electrical charge is applied to its droplet at the moment of detachment.
These charged droplets pass through a powerful electric field generated by high-voltage deflection plates. The charge causes the droplet to be electrostatically deflected into a specific collection tube, while unwanted, uncharged droplets are discarded. This physical separation allows researchers to isolate and purify specific cell populations for further study with high accuracy and speed.
Decoding the Results
The numerical data collected by the electronics system—FSC, SSC, and multiple fluorescence intensities—is translated into visual representations for interpretation. Two common graphical formats are used: histograms and dot plots. A histogram is a single-parameter plot that shows the frequency of cells along a single axis of measurement, such as the number of cells that have a certain level of a single fluorescent marker.
More complex analysis uses two-parameter dot plots, where each dot represents a single cell. Its position is determined by the intensity of two measured parameters, such as size (FSC) versus internal complexity (SSC). These plots allow researchers to visualize distinct populations within the sample, as clusters of dots form based on shared characteristics. For example, in a blood sample, lymphocytes, monocytes, and granulocytes form three distinct clusters on an FSC versus SSC plot.
The process of “gating” is then applied, which involves drawing a boundary around a specific cluster of cells on the dot plot to isolate that particular population for further analysis. By sequentially applying gates across multiple plots, researchers can filter the data to identify and quantify specific subsets, such as a T-cell (gated by size) that also expresses two specific surface proteins (gated by fluorescence). This visual, iterative analysis allows the high-speed electronic signals to yield meaningful biological conclusions.