Flow cytometry is a powerful laboratory technique used to rapidly analyze the characteristics of individual cells or particles as they flow in a fluid stream. This method collects detailed, multiparameter data from thousands of cells each second, allowing for an understanding of cell populations by examining their physical and fluorescent properties.
Preparing Samples for Analysis
Before analysis, cells undergo careful preparation to create a single-cell suspension. Cells are typically sourced from biological materials like blood, tissue, or cell cultures, and must be dissociated into individual cells to prevent clumping. For solid tissues, this often involves mechanical disruption or enzymatic digestion to separate cells.
A key step involves staining cells with fluorescent dyes or antibodies, known as fluorochromes. These specialized molecules bind to specific cellular components or surface markers, making particular features visible and distinguishable when they interact with a laser inside the instrument. Different fluorochromes emit light at distinct wavelengths, allowing simultaneous study of multiple cell characteristics.
The Journey Through the Cytometer
Once prepared, the sample enters the flow cytometer, which uses fluidics, optics, and electronics. The fluidics system is responsible for precisely delivering cells, one by one, to the laser interrogation point. This occurs through hydrodynamic focusing, where a sample stream is injected into a faster-moving sheath fluid. The sheath fluid surrounds and compresses the sample, forcing cells into a single-file line through a narrow channel.
As each cell travels through this focused stream, it encounters one or more laser beams, the optical system. Lasers use coherent, monochromatic, and energetic light, ensuring uniform cell illumination. When a cell intercepts the laser, it scatters light in various directions. Forward scatter (FSC) is light scattered along the laser’s path, providing information primarily about cell size. Side scatter (SSC) measures light scattered at a 90-degree angle, indicating the cell’s internal complexity or granularity, such as organelles.
Additionally, fluorochromes attached to cells emit fluorescent light when excited by the laser. A system of lenses and filters then collects this scattered and emitted light. Dichroic mirrors and bandpass filters direct specific wavelengths to their detectors, ensuring accurate measurement from each unique fluorochrome.
Finally, the electronics system converts these light signals into digital data. Photomultiplier tubes (PMTs) or avalanche photodiodes (APDs) detect collected light, transforming photons into electrical pulses. These signals are then amplified and digitized, allowing a computer to process and store the information for each individual cell.
Understanding the Data
After signals are collected, they are translated into meaningful data points, displayed on scatter plots. On these plots, each dot represents an individual cell, positioned by measured parameters like size (FSC), granularity (SSC), and fluorescence intensity from various stains. This allows researchers to identify distinct cell populations within a heterogeneous sample.
Gating involves drawing regions around specific clusters of dots on these plots. This selects and isolates specific cell populations for further analysis based on their unique characteristics. Gating excludes unwanted events like debris or clumps, ensuring analysis of only viable, single cells. Through this analysis, researchers quantify percentages of different cell types, measure marker expression levels, and identify rare cell populations within a sample.
Where Flow Cytometry is Applied
Flow cytometry is a versatile technique applied across various scientific and medical fields. In immunology, it is used to diagnose immune deficiencies and monitor diseases like HIV by counting specific immune cell populations, such as CD4+ T cells. It also helps study different immune cell subsets.
In cancer research, flow cytometry is instrumental in diagnosing and classifying various types of leukemia and lymphoma based on distinct cell surface markers. It also monitors the effectiveness of cancer treatments by assessing changes in tumor cell populations.
Flow cytometry contributes to fundamental cell biology research by analyzing the cell cycle, programmed cell death (apoptosis), and cell proliferation. It also applies to microbiology and environmental science, analyzing and characterizing different types of bacteria or phytoplankton in water samples.