Flow cytometry is a technique used to analyze individual cells or particles as they flow in a single stream. Lasers are a fundamental component, illuminating and interacting with these cells. This allows researchers to gather detailed information about cell characteristics, making lasers indispensable for understanding cellular populations.
The Role of Lasers in Flow Cytometry
Lasers are central to flow cytometry because they produce highly focused, monochromatic light. This light consists of a single, precise wavelength, ideal for interacting with cells in a controlled manner. As cells pass through the laser beam, two primary interactions occur, providing distinct types of information.
The first interaction is light scatter, which provides insights into a cell’s physical properties. When laser light hits a cell, it scatters in different directions. Forward scatter (FSC) is detected along the same axis as the laser beam and relates to cell size. Side scatter (SSC) is detected at a 90-degree angle and provides information about the cell’s internal complexity or granularity, such as cytoplasmic granules or the nucleus. These measurements are fundamental for distinguishing cell populations based on their morphology.
The second interaction involves the excitation of fluorescent dyes, known as fluorochromes, attached to the cells. When a fluorochrome absorbs light from the laser at a specific excitation wavelength, its electrons temporarily move to a higher energy state. As these electrons return to their ground state, they emit light at a longer, less energetic wavelength, a process called fluorescence. This emitted fluorescent light has a different wavelength from the original laser light, allowing for specific detection of labeled cellular components.
Common Laser Types and Their Characteristics
Flow cytometers employ various lasers, each emitting light at a specific wavelength to excite different fluorochromes. The choice of laser depends on the fluorescent markers used in an experiment.
Blue lasers, typically operating at 488 nm, are widely used and often considered a standard due to their ability to excite common fluorochromes. These include fluorescein isothiocyanate (FITC) and phycoerythrin (PE), both frequently used in cell analysis. Solid-state lasers have largely replaced older argon-ion lasers for this wavelength.
Red lasers, commonly emitting at 633 nm, 635 nm, or 640 nm, are also prevalent. They are effective for exciting far-red dyes such as Allophycocyanin (APC) and its tandem conjugates. These lasers extend the range of detectable fluorochromes, allowing for more complex multi-color experiments.
Violet lasers, often 405 nm diode lasers, have become increasingly common. These lasers are particularly useful for exciting dyes like DAPI (4′,6-diamidino-2-phenylindole), which binds to DNA, or Pacific Blue, a common fluorochrome for immunophenotyping. Violet laser diodes are smaller and less expensive than older krypton-ion lasers, making them a practical choice for many instruments.
Other lasers, while less common as primary sources, contribute to advanced flow cytometry. Yellow-green lasers, typically at 561 nm, are efficient at exciting PE and its tandem dyes. Ultraviolet (UV) lasers, typically at 355 nm, are used for specialized dyes like those in the Brilliant Ultraviolet (BUV) series or for DNA content analysis using dyes like DAPI.
How Lasers Generate Data Signals
After the laser beam interacts with cells, the emitted light signals are collected and processed. Both scattered light and fluorescent light are directed towards specialized optical detectors. These detectors capture photons, which are then converted into electrical pulses.
Optical filters separate different wavelengths of light. Bandpass filters are positioned in front of detectors to allow only a specific, narrow range of wavelengths to pass through. This ensures signals from different fluorochromes are measured independently, preventing overlapping signals from interfering.
The filtered light signals then reach photodetectors, most commonly photomultiplier tubes (PMTs). When photons strike the photocathode within a PMT, they eject electrons. These electrons are amplified, generating a stronger electrical signal. The intensity of these electrical pulses is directly proportional to the amount of light detected, reflecting the quantity of fluorescent molecules or the degree of light scatter.
These electrical signals are digitized and processed by the instrument’s software. The software converts analog electrical pulses into digital values, which are then displayed as data plots, such as histograms or scatter plots. Histograms show the intensity of a single parameter, while scatter plots display the relationship between two or more parameters, allowing researchers to analyze different cell populations.
Matching Lasers to Specific Research Needs
Researchers select lasers and their corresponding wavelengths based on the biological questions they aim to answer and the fluorescent markers they employ. This tailored approach allows for detailed analysis of cellular characteristics and functions.
For instance, a blue laser (488 nm) is frequently chosen for immunophenotyping experiments, which involve identifying different immune cell types using common fluorescent antibodies. When analyzing DNA content or cell cycle progression, a violet laser (405 nm) is often paired with DNA-binding dyes like DAPI or DyeCycle Violet. The ability to combine specific lasers with appropriate fluorochromes enables complex, multi-parameter analysis of cells.