Analyzing individual cells is a foundational practice in modern biological science and clinical diagnostics, allowing researchers to move beyond bulk sample measurements to understand the distinct properties of every element within a population. This single-cell resolution is necessary for accurately characterizing complex biological systems like the immune system or tumor microenvironments. The CytoFLEX flow cytometer is a contemporary instrument designed for detailed, high-throughput analysis in a compact format. It provides researchers with a platform for rapidly measuring multiple characteristics of thousands of cells per second, integrating advanced optical technology into an accessible benchtop system.
Understanding Flow Cytometry
Flow cytometry is a technique that uses fluid dynamics, optics, and electronics to measure various physical and chemical characteristics of particles or cells suspended in a liquid. The process begins with the fluidics system, which employs hydrodynamic focusing. The sample stream containing the cells is injected into a faster-moving sheath fluid, which confines the cells to the center of the stream. This forces them to pass through the laser interrogation point one at a time. This single-file movement ensures the instrument measures each cell individually.
As each cell travels through the focused laser beam, it scatters the incident light and, if stained with fluorescent dyes, emits light. Detectors collect the scattered light in two primary ways. Forward Scatter (FSC) is measured along the laser’s axis and indicates the cell’s relative size. Side Scatter (SSC) is collected at a 90-degree angle to the laser beam and correlates with the cell’s internal complexity or granularity.
The fluorescent light is collected and separated into specific wavelengths by filters. Biological components, like surface proteins, are tagged with fluorescent molecules called fluorochromes. When the laser excites these fluorochromes, they release energy as light. The intensity of this emitted light allows researchers to quantify the amount of a specific target molecule present on or within the cell.
Defining the CytoFLEX Architecture
The CytoFLEX platform incorporates technological innovations that enhance its sensitivity and reduce its physical footprint compared to traditional instruments. Its compact, benchtop design uses integrated optics and a simplified fluidics system that maintains optical alignment without requiring a large, complex frame. The system replaces bulkier optical components with more efficient light management strategies.
A significant architectural feature is the use of fiber optics to deliver excitation light to the flow cell and collected emission light to the detectors. The instrument also features a patented Wavelength-Division-Multiplexing (WDM) filter system. This WDM system uses reflective optics and bandpass filters to efficiently separate the collected light into its constituent wavelengths, eliminating the need for multiple dichroic mirrors. Minimizing light loss contributes to the instrument’s sensitivity.
The CytoFLEX utilizes Avalanche Photodiode (APD) detectors instead of traditional Photomultiplier Tubes (PMTs) for fluorescence and side scatter detection. APDs offer a high quantum efficiency, especially for light in the far-red and infrared wavelengths, where they can exceed 80% efficiency. This high efficiency translates into a better signal-to-noise ratio, allowing the instrument to detect dimly stained cell populations with greater resolution. The combination of APD detectors and the WDM filter system results in sensitive signal collection and reduces the complexity of spectral compensation, which is the mathematical correction for overlapping fluorescence signals.
Practical Applications of the CytoFLEX
The multi-parameter capability of the CytoFLEX has expanded its utility across various research fields.
Immunology and Monitoring
In immunology, the instrument is employed for deep immune phenotyping, allowing for the simultaneous identification and quantification of complex T-cell subsets based on surface markers. This capability is used for long-term immune monitoring in patients undergoing treatment for infectious diseases or cancer, tracking changes in specific immune cell populations over time.
Cancer Research and Rare Cells
In cancer research, the cytometer analyzes rare cell populations, such as circulating tumor cells (CTCs) found in the peripheral blood. The enhanced resolution allows researchers to detect these low-frequency events, which are used as biomarkers to monitor disease progression or treatment effectiveness. The platform’s ability to analyze particles smaller than 200 nanometers using Violet Side Scatter (VSSC) also makes it suitable for studying extracellular vesicles (EVs). Researchers characterize EVs derived from tumor cells, which carry molecular cargo like DNA or protein markers that reflect the state of the parent tumor.
Environmental Analysis
Beyond the biomedical field, the instrument is applied in environmental and marine biology for the analysis of small microorganisms. The high sensitivity for sub-micron particles means it can effectively analyze bacteria, phytoplankton, or other nano-sized biological entities in water samples. By combining size and granularity measurements with fluorescent probes, researchers can rapidly assess the composition and health of microbial communities in aquatic ecosystems.
Facilitating High-Parameter Analysis
The architectural design of the CytoFLEX enables high-parameter analysis, which is the simultaneous measurement of a large number of distinct markers on a single cell. Standard configurations measure up to 13 fluorescent colors, and expanded models analyze over 20 parameters using up to six spatially separated lasers. This multiplexing provides researchers with a comprehensive snapshot of cellular biology.
High-parameter analysis allows for a deeper understanding of cellular heterogeneity, as multiple characteristics—such as a cell’s size, granularity, and expression of proteins—are correlated for every event. The data generated from a single experiment involves hundreds of thousands of individual cells, each described by numerous data points. This complexity necessitates the use of specialized software, such as the CytExpert platform, which manages the datasets and performs the required spectral compensation to separate the overlapping signals. The resulting high-dimensional data allows for the discovery of previously uncharacterized cell populations and subtle changes in cellular states.