Flow cytometry is a technology designed to analyze the physical and chemical characteristics of thousands of individual cells or particles suspended in a fluid stream at high speed. This method allows scientists to gather detailed information about a cell population by measuring how each cell interacts with a focused beam of light, typically a laser. The instrument works by simultaneously assessing multiple parameters for every cell.
The core principle involves shining a laser onto a cell that has been chemically prepared and then collecting the light that is scattered and emitted. By converting these optical signals into electronic data, the flow cytometer can rapidly quantify properties such as a cell’s size, internal complexity, and the presence or absence of specific molecular markers. It is capable of analyzing upwards of 10,000 cells per second.
Preparing Cells for Analysis
Successful flow cytometry begins with sample preparation, ensuring cells can be analyzed one at a time. The fundamental requirement is creating a single-cell suspension, meaning all cells must be free-floating and not clumped together. Clumps can obstruct the fluid path or be incorrectly registered as a single, large event, leading to inaccurate measurements.
For solid tissues, a process of disaggregation, often using enzymes or mechanical methods, is required to separate the cells. Once in suspension, the next step is fluorescent labeling, or “staining.” Researchers use fluorochrome-conjugated antibodies, which bind to a specific target on or inside the cell and emit light when excited by the laser.
This molecular tagging allows the machine to identify specific cell components, such as surface proteins or intracellular molecules. For example, an antibody targeting the CD4 protein on a T-cell is linked to a dye that fluoresces a particular color, measuring the amount of CD4 protein present. Filtering the final suspension through a fine mesh (typically 35 to 50 microns) removes any remaining aggregates before the sample is run.
How the Flow Cytometer Works
The flow cytometer is comprised of three integrated systems—fluidics, optics, and electronics—that execute the cell-by-cell analysis. The fluidics system guides the cells to the interrogation point where they meet the laser beam. This system uses hydrodynamic focusing, where the sample stream containing the cells is injected into a faster-moving stream of sheath fluid.
The force of the outer sheath fluid compresses the inner sample stream, aligning the cells into a narrow, single-file line. This ensures that only one cell passes through the focus of the laser at any given moment, which is essential for accurate, individual measurement. Once aligned, the cells enter the optics system, where they are illuminated by one or more lasers.
When a cell passes through the laser, two types of light signals are generated and collected by detectors:
Light scatter, which provides information about the cell’s physical properties. Forward Scatter (FSC) correlates with the cell’s relative size, while Side Scatter (SSC) relates to the cell’s internal complexity and granularity.
Fluorescence emission, which is light emitted by the excited fluorophores attached to the cell’s molecules.
A system of mirrors and optical filters directs the emitted light to specific detectors, allowing the instrument to distinguish between the various colors produced by different fluorescent tags. The electronics system converts these collected light signals, which are proportional to the physical and fluorescent properties, into digital data points. Each individual cell, or “event,” is represented by a set of numbers corresponding to its measured size, granularity, and the intensity of every fluorescent marker.
Understanding the Data Output
The data collected by the flow cytometer is translated into visual representations, primarily scatter plots and histograms, which organize individual cell events. Scatter plots display two measured parameters on the X and Y axes, with each dot representing a single cell.
A common scatter plot pairs FSC (size) against SSC (granularity), enabling the visual separation of different cell types, such as lymphocytes from monocytes and granulocytes in a blood sample. By drawing a graphical boundary, or “gate,” around a distinct cluster of dots, researchers can isolate a specific cell population for subsequent analysis.
Histograms display the frequency distribution of a single parameter, such as the fluorescence intensity of a molecular marker. The X-axis shows the signal intensity, and the Y-axis represents the number of cells counted. A shift in the peak of the histogram to the right indicates an increase in the expression of that specific molecule.
Real-World Uses of Flow Cytometry
Flow cytometry’s capacity to rapidly analyze multiple cellular properties makes it a powerful method across various fields of medicine and research. In clinical diagnostics, it is used for immunophenotyping, which involves identifying and counting different types of immune cells. This is relevant for monitoring HIV infection by quantifying the number of CD4+ T-cells.
The technology aids in diagnosing and classifying blood cancers, such as leukemia and lymphoma. By using panels of fluorescent antibodies, pathologists identify abnormal white blood cell populations based on the unique combination of proteins expressed on their surface, aiding in subtype classification and guiding treatment strategies.
In basic biological research, flow cytometry is used to study cell function. Scientists apply the method to track cell proliferation, assess the cell cycle, and detect apoptosis (programmed cell death). Specific fluorescent dyes can monitor DNA content or label actively dividing cells, providing quantitative data on how a cell population responds to experimental conditions.