Flow cytometry is a laser-based technology that rapidly measures physical and chemical properties of thousands of individual cells per second as they flow in a single-file stream past optical detectors. It enables multiparametric analysis of cell populations at single-cell resolution.
Principles
Cells in suspension are hydrodynamically focused into a narrow stream, ensuring they pass individually through the interrogation point where one or more laser beams intersect the stream. As each cell passes through the laser, it scatters light and emits fluorescence if labeled with fluorophores. The scattered and emitted light is collected by detectors, converted to electronic signals, and digitized for analysis.
Forward and Side Scatter
Forward scatter measures light scattered at small angles, correlating with cell size. Larger cells scatter more light in the forward direction. Side scatter measures light scattered at 90 degrees, reflecting cell granularity and internal complexity. Neutrophils with many granules have high side scatter, while lymphocytes have low side scatter. Together, FSC and SSC can distinguish major leukocyte populations in blood without any staining.
Fluorescence Detection
Fluorescence is emitted when fluorophores excited by the laser return to their ground state. Cells are typically stained with fluorescently conjugated antibodies targeting specific surface or intracellular markers, similar to antibody-based detection in ELISA. Each fluorophore has characteristic excitation and emission spectra. Flow cytometers use dichroic mirrors and bandpass filters to separate fluorescence into distinct detector channels, each measuring a specific wavelength range.
Fluorophores
Common fluorophores include FITC, PE, APC, PerCP, and Alexa Fluor dyes. Tandem dyes such as PE-Cy7 combine two fluorophores, where energy transfer from the donor to acceptor produces a large Stokes shift. Modern cytometers can measure 12 to 50 parameters simultaneously using multiple lasers and detectors.
Compensation corrects for spectral overlap between fluorophores. When the emission spectrum of one fluorophore spills into the detector of another, the contribution must be mathematically subtracted. Compensation controls using single-stained samples are essential for accurate multicolor analysis.
Cell Sorting
Fluorescence-activated cell sorting physically separates cells based on their measured properties. The sorter uses a vibrating nozzle to generate droplets containing single cells. Droplets are electrically charged based on the cell’s measured characteristics and deflected by electrostatic plates into collection tubes. Sorting purity typically exceeds 95%. Aseptic sorting is possible for downstream culture or functional assays.
Applications
Immunophenotyping identifies and quantifies immune cell subsets using panels of lineage-specific markers. CD4 T cell counts in HIV monitoring are a clinical application of flow cytometry. Cell cycle analysis measures DNA content using propidium iodide or DAPI staining, identifying G1, S, G2, and M phase populations.
Apoptosis assays detect phosphatidylserine exposure with annexin V, membrane permeability with propidium iodide or 7-AAD, and caspase activation with fluorescent inhibitors. Intracellular cytokine staining measures cytokine production in activated T cells. Phospho-flow measures phosphorylation of signaling proteins at single-cell resolution, analogous to Western blot analysis. Fluorescent protein expression from reporters such as GFP can be measured without antibody staining.
Spectral Flow Cytometry
Spectral flow cytometry uses a diffraction grating or prism to capture the full emission spectrum of each cell across many detectors, rather than measuring in discrete channels. Spectral unmixing algorithms deconvolve the contribution of each fluorophore. This approach accommodates more fluorophores with similar spectra and simplifies panel design by reducing compensation requirements.
Mass Cytometry
Mass cytometry uses antibodies conjugated to stable heavy metal isotopes rather than fluorophores. Cells are introduced into an inductively coupled plasma mass spectrometer, which atomizes and ionizes the metals. Each isotope mass is measured by time-of-flight mass spectrometry. Mass cytometry can measure 40 or more parameters without spectral overlap or autofluorescence. However, cells are destroyed during analysis and cannot be recovered. The technology has lower throughput than conventional flow cytometry.
