A flow cytometer rapidly analyzes the physical and chemical characteristics of individual cells or particles suspended in a fluid. This technology allows researchers to quantitatively measure numerous features for tens of thousands of cells every second. By processing cells one at a time, the cytometer gathers high-resolution data that reveals the composition and function of a complex cell population. This approach provides an automated, high-throughput alternative to traditional microscopy for analyzing cellular components and specific molecular markers. The entire process relies on three interconnected systems: fluidics, optics, and electronics, which transform a cell suspension into usable digital data.
The Role of Fluidics in Cell Alignment
The first step in analyzing a cell sample requires precisely controlling the movement of particles through the instrument’s core. This is achieved by hydrodynamic focusing, which ensures that each cell passes individually through the measurement point. The fluidics system introduces the cell sample into a wider stream of surrounding liquid, known as the sheath fluid. The sheath fluid is pumped at a higher pressure than the sample, causing it to compress the inner sample stream into a narrow core.
This differential pressure forces the cells into a single-file line as they travel toward the laser beam. The process relies on laminar flow, meaning the two fluids—the sample core and the sheath fluid—flow in parallel layers without mixing. This stable, focused stream prevents multiple cells from passing through the laser simultaneously, which would result in inaccurate measurements. The flow cell nozzle constricts the stream, aligning the cells to ensure optimal exposure to the light source before they are analyzed.
Illuminating and Measuring Cells with Optics
Once the cells are aligned, they travel through the interrogation point, where they intersect with one or more focused laser beams. This interaction causes the light to scatter and, if the cells are stained with fluorescent dyes, to emit light at different wavelengths. The scattered light is collected by specialized detectors and provides fundamental information about the cell’s physical structure.
Scatter Measurements
Two primary scatter measurements are taken: Forward Scatter (FSC) and Side Scatter (SSC). The FSC detector is positioned along the same axis as the laser beam, measuring the light diffracted around the cell, which correlates directly with the cell’s relative size or volume. The SSC detector is placed perpendicular to the laser path, capturing light that is refracted by internal cellular structures. This SSC signal provides information about the cell’s internal complexity, such as the presence of a granular cytoplasm or a lobed nucleus.
Fluorescence Detection
In addition to scatter, the optics system measures fluorescence, which is light emitted by specific fluorescent molecules, or fluorochromes, attached to the cells. These fluorochromes are typically coupled to antibodies that bind to distinct cellular components, such as surface proteins or internal DNA. When the laser excites these dyes, they emit light at a longer, characteristic wavelength. A series of dichroic mirrors and optical filters precisely direct this emitted light to separate photodetectors. The mirrors selectively reflect specific wavelengths while allowing others to pass through, effectively separating the different fluorescent signals into distinct channels for individual measurement.
Converting Light Pulses into Digital Data
The final stage of the flow cytometer involves converting the light signals captured by the detectors into a format a computer can process. Photodetectors, such as photomultiplier tubes (PMTs) for scattered and fluorescent light, convert the photons into a proportional electrical current. As a cell passes through the laser, a transient electrical signal, known as a voltage pulse, is generated. The intensity of the light hitting the detector dictates the magnitude of this voltage pulse.
The electronics system then amplifies this weak analog signal to a measurable level. This amplified signal is immediately sent to an Analog-to-Digital Converter (ADC), which samples the pulse and converts the continuous analog voltage into a specific digital number. Modern cytometers often use high-resolution ADCs, such as 16-bit converters, to provide a wide dynamic range and finely resolve differences in light intensity. For each single cell, the system records the peak height, area, and width of the pulse for every measured parameter, assigning a unique data point.
This digitized data is then stored in a file format for subsequent analysis, where it is visualized. Histograms are often used to display the distribution of a single parameter, such as the intensity of a specific fluorescent marker, across the cell population. More commonly, researchers use two-dimensional scatter plots, which display two parameters simultaneously, such as FSC versus SSC, or two different fluorescent markers. These plots allow for the visual separation of distinct cell populations based on their unique light scatter and fluorescence profiles.