Analyzing complex biological samples requires examining individual cells rather than bulk populations. Flow cytometry is a powerful laboratory technology that rapidly analyzes the physical and chemical characteristics of thousands of cells suspended in a fluid stream. The technique uses focused lasers and specialized detectors to gather data on each cell one at a time. Fluorescence-Activated Cell Sorting (FACS) represents a significant advancement by adding the capability to physically separate the cells of interest.
Fluorescence Activated Cell Sorting Defined
FACS is a specialized application of flow cytometry. Standard flow cytometry is purely an analytical tool; it counts cells and provides extensive data on cell properties but cannot isolate them, sending the entire sample to waste. FACS incorporates the cell sorting step, which is the “Activated Cell Sorting” component of the acronym. This functionality allows researchers to physically separate specific cell types from a heterogeneous mixture based on the collected data. The ability to isolate a pure population of cells for further experimentation makes FACS invaluable when a highly purified sample is necessary.
Principles of Cell Measurement
The initial phase of any FACS procedure involves the precise measurement of each cell’s properties, utilizing the fundamental principles of flow cytometry. This measurement phase begins with the fluidics system and hydrodynamic focusing. The cell sample is injected into the center of a surrounding sheath fluid, forcing the cells to line up single-file toward the laser interrogation point. This alignment ensures that only one cell is analyzed at a time, allowing for accurate, high-speed data collection.
Once aligned, individual cells pass through focused laser beams. The laser light generates two types of signals: scattered light and emitted fluorescent light. Detectors positioned in line with the laser measure forward scatter, estimating the cell’s size. Other detectors positioned perpendicularly measure side scatter, which provides information about the cell’s internal complexity or granularity.
For the “Fluorescence” component, cells are pre-treated with antibodies tagged with fluorochromes that bind to specific cellular markers. When the laser excites these fluorochromes, they emit light at a specific, longer wavelength. Additional detectors measure this emitted light, identifying the presence and quantity of specific molecules, such as surface proteins. These combined measurements—size, granularity, and specific fluorescence—create a comprehensive profile for every cell passing through the instrument.
The Mechanism of Cell Sorting
The process transitions to sorting immediately after the cell passes the laser and its characteristics are recorded. Based on pre-set criteria, the instrument’s software rapidly decides if the cell is a desired type. A piezoelectric crystal vibrates the fluid stream, causing it to break into individual, uniform droplets. The vibration timing is precisely controlled so that each droplet ideally contains only a single cell.
Just before the stream breaks, an electrical charging ring is activated if the droplet contains a cell of interest. This ring applies a positive or negative electrostatic charge to that specific droplet. Droplets containing unwanted cells remain uncharged, or are given an opposite charge for waste collection.
The charged droplets then fall through a strong electrostatic field created by two high-voltage deflection plates. The charged droplets are attracted toward the plate with the opposite charge, deflecting them into one of several collection tubes. Uncharged droplets pass straight into a central waste container. This system allows for the isolation of multiple distinct cell populations simultaneously, resulting in highly purified samples that can reach purities greater than 99%.
Clinical and Research Applications
The combination of detailed analysis and physical separation makes FACS an indispensable tool in medicine and biological research.
Clinical Diagnostics
In clinical diagnostics, immunophenotyping is a common application. This involves identifying and counting specific immune cell subsets in patient samples. FACS is used to:
- Diagnose and monitor blood cancers, such as leukemia and lymphoma, by detecting abnormal cell surface markers.
- Track the progression of HIV by quantifying the number of specific T-cells.
Research Applications
In research, FACS is widely used for isolating rare and specialized cell types for further study.
- Researchers purify stem cells from mixed populations to investigate their potential for regenerative therapies.
- In oncology, FACS isolates tumor cells for detailed molecular profiling, helping scientists understand cancer progression.
- The precision sorting capability accelerates the study of cell signaling pathways and the development of new vaccines.