As an important tool in modern biomedical research and clinical diagnostics, flow cytometry integrates technologies from multiple disciplines—including optics, electronics, fluid mechanics, and computer science—and enables high-speed, multiparameter quantitative analysis and sorting of cells or biological particles. Since its development in the 1970s, flow cytometry has played an irreplaceable role in fields such as immunology, hematology, oncology, and cell biology.

1. The basic principle of flow cytometry 
　　The fundamental principles of flow cytometry can be traced back to the 1960s. After more than half a century of development, it is now capable of simultaneously measuring dozens of parameters—such as cell size, granularity, and fluorescence intensity—at a rate of tens of thousands of cells per second, providing powerful instrumental support for life science research. 
　　The core function of a flow cytometer is the rapid, multiparameter quantitative analysis and sorting of single cells or biological particles suspended in a fluid. Its working mechanism is based on three key technologies: hydrodynamic focusing, optical detection, and electronic signal processing. In the hydrodynamic focusing process, the cell sample is first prepared into a single-cell suspension. Then, within the flow chamber of the flow cytometer, the cells are surrounded by sheath fluid. By precisely controlling the flow rates and pressures of both the sheath fluid and the sample stream, the cells are guided to pass sequentially through the detection zone in a single-file manner, enclosed by the sheath fluid. This "single-cell flow" technology ensures that each cell can be individually detected, thereby avoiding signal interference caused by overlapping cells passing through the laser beam. Hydrodynamic focusing technology is crucial for the flow cytometer to achieve high-throughput single-cell analysis, enabling the instrument to analyze thousands to tens of thousands of cells per second.

The working principle of a flow cytometer

　　When cells pass individually through the detection area, they are illuminated by one or more laser beams, generating various types of optical signals. These signals primarily include scattered light and fluorescence. Scattered light is further divided into forward scatter (FSC) and side scatter (SSC). The former is correlated with the cell’s size and surface area, while the latter reflects the cell’s internal structure and particle complexity. By simultaneously measuring FSC and SSC, it is possible to distinguish between different populations of cells—for example, in blood samples, to differentiate between lymphocytes, monocytes, and granulocytes. 
　　The fluorescence signal originates either from the cells themselves or from fluorescent labels that are bound to the cells. When a laser beam excites the fluorescent dye, these dyes absorb light energy at a specific wavelength and re-emit light at a longer wavelength. A flow cytometer can simultaneously detect multiple fluorescence signals. By using multi-color labeling of cells, it is possible to obtain multidimensional information about cell surface markers, intracellular molecules, and the functional state of cells. Fluorescence detection boasts extremely high sensitivity, enabling it to detect signals from as few as several thousand fluorescent molecules on a single cell. 
　　After the optical signal is collected, it is guided through a series of optical filters and lenses to the corresponding photodetector, where it is converted into an electrical signal. These weak electrical signals are then amplified by an amplifier and subsequently converted into digital signals by an analog-to-digital converter, before being transmitted to a computer for storage and analysis. The specialized software equipped in modern flow cytometers not only displays data in real time but also enables sophisticated post-analysis tasks, such as population gating, cell cycle analysis, and proliferation analysis.

The main types of signals detected by flow cytometry and their biological significance are summarized as follows:

　　It is worth noting that, with advances in technology, flow cytometry has evolved from traditional fluorescence-based flow cytometry to a diversified technological platform encompassing multiple detection principles. Emerging flow cytometry techniques such as Raman scattering flow cytometry, mass cytometry (CyTOF), and imaging flow cytometry are continually broadening researchers' horizons. In particular, the optical flow cytometer based on Optical Time-Stretch Imaging (OTS) technology boasts advantages including ultra-short exposure times, high spatial resolution, real-time high-throughput analysis, and continuous real-time imaging. Theoretical throughput can reach the million-level, making it a cutting-edge development direction in flow cytometry.

2. System Components of a Flow Cytometer 
　　As a highly integrated and sophisticated analytical instrument, the flow cytometer’s system architecture incorporates a variety of engineering technologies, primarily comprising four core components: the fluidics system, the optical system, the detection system, and the data analysis system (detection and analysis system). These subsystems work in concert to complete the complex analytical process—from sample input to data output. 
　　The flow system is the fundamental supporting component of a flow cytometer, responsible for sequentially delivering the cells to be analyzed from the cell suspension into the laser detection zone. This system consists of components such as a sample injection device, sheath fluid container, flow chamber, and waste liquid collection device. 
　　The optical system is the “eyes” of the flow cytometer, performing the dual function of exciting the sample and collecting optical signals. Its core components include lasers, optical lens assemblies, and detectors. As the excitation light source, the laser’s wavelength and power directly affect detection sensitivity and fluorescent labeling. Modern high-end flow cytometers typically employ a combination of four laser wavelengths—typically 405 nm, 488 nm, 561 nm, and 640 nm. The laser beam is focused onto the detection point in the flow chamber through a series of lenses, forming an elliptical or flattened linear spot. When cells pass through the laser beam, the scattered light and fluorescent signals they generate are collected by focusing lenses and then directed to the corresponding detectors. Spectral separation devices—such as dichroic mirrors and bandpass filters—are responsible for routing optical signals of different wavelengths into specific detection channels. In terms of detectors, traditional photomultiplier tubes (PMTs) are gradually being partially replaced by newer devices like silicon photomultipliers (SiPMs), which offer higher sensitivity and lower noise levels.

System Components of a Flow Cytometer 
 

　　The detection system serves as a signal conversion and amplification unit, comprising primarily an amplifier, an analog-to-digital converter (ADC), and a real-time processing unit. The electronic system coordinates the timing of all instrument components, controls laser triggering, fluid flow stability, and detection synchronization—it acts as the “nervous system” of the flow cytometer. 
　　The data analysis system serves as the “brain” of the flow cytometer, responsible for data storage, display, and analysis. As the number of flow cytometry parameters increases—modern instruments can simultaneously detect over 50 parameters—and experimental complexity rises, data processing systems face significant challenges. In recent years, the emergence of full-spectrum flow cytometers has placed even higher demands on data analysis. These instruments capture the complete emission spectra of each fluorescent dye and use algorithms to parse and determine the relative contribution of each dye.

The functions and performance indicators of each component of the flow cytometer are summarized as follows:

　　The technological advancements in flow cytometry are closely linked to innovations in its system components. Take, for example, the Optical Time Stretch Imaging Flow Cytometer (OTS-IFC). This novel flow cytometer combines optical time-stretch technology with a microfluidic system, enabling high-speed imaging flow cytometry with ultra-short exposure times—on the order of picoseconds—and achieving a theoretical throughput of up to millions of cells per second. It provides a completely new solution for applications that have been difficult to realize with conventional flow cytometers, such as rare cell identification and studies on cellular morphodynamics.

3. Typical lasers used for streaming 
　　The lasers used in flow cytometers are subject to extremely high requirements. In practical applications, the settings of laser parameters need to be optimized and adjusted according to the sample type, labeling strategy, and detection targets (such as surface markers versus intracellular factors). With technological advancements, the laser systems in flow cytometers are evolving toward higher power stability, broader wavelength coverage, and more compact, modular designs. 
　　Modern high-end flow cytometers commonly employ multi-channel laser configurations to meet the demands of complex experiments, featuring systems with four or more laser wavelengths—such as the combination of 349 nm/375 nm/405 nm/488 nm/561 nm/638 nm/808 nm—which support analysis of 21 or more colors, offer comprehensive spectral coverage, and minimize crosstalk. 
　　CNI can provide, in bulk, typical single-wavelength and multi-wavelength flow-through light sources. We also offer customized wavelengths, integration, and beam shaping and homogenization tailored to customer requirements. For more details, please visit www.cnilaser.com.

Single-wavelength flow light source	Multi-wavelength combined flow-through light source
261, 320, 349, 355, 375, 405, 488, 532, 561, 638, 640, 808 nm... and more—customizable.	349/405/488/640/561 combination, 405/488/640/561 combination... and so on—customizable.

4. Applications of Flow Cytometry 
　　Thanks to its unique advantages—high throughput, multiparameter analysis, and high sensitivity—flow cytometry has now permeated numerous fields in life science research and clinical applications. From fundamental cell biology studies to cutting-edge precision medicine practices, flow cytometers play an irreplaceable role. As the technology continues to advance and innovate, the scope of flow cytometry applications keeps expanding, providing powerful instrumental support for scientific discovery and disease diagnosis and treatment.

Flow cytometry for immunophenotyping of whole human blood 
 

The typical applications of flow cytometry in clinical diagnosis are summarized as follows:

With technological advancements, the application scope of flow cytometry continues to expand. For example, mass cytometry (CyTOF) can simultaneously detect more than 40 parameters, significantly enhancing the capacity for systems immunology research. Meanwhile, imaging flow cytometry integrates morphological information, providing a new dimension for studying cellular functions. In the era of precision medicine, flow cytometry will continue to play a pivotal role in molecular subtyping of diseases, predicting treatment responses, and developing personalized therapeutic strategies.