A Fluorescence Activated Cell Sorter, commonly known as FACS, is a sophisticated laboratory instrument for analyzing and separating cells. It allows scientists to examine millions of cells quickly, identifying them by unique characteristics. Its precision and speed make it a valuable tool for biological research and medical applications. FACS sorts cells based on specific properties, providing pure populations for further study, enhancing understanding of cellular processes.
How Cells Are Identified and Separated
FACS operation begins with labeling cells using fluorescent tags. Researchers attach fluorescent dyes (fluorochromes) to antibodies that bind to molecules on the cell surface or inside. These tags highlight specific cellular components, allowing the instrument to recognize them. This labeling differentiates cell types within a mixed population based on their molecular signatures.
Labeled cells are suspended in fluid and introduced into the cytometer’s fluidic system. Hydrodynamic focusing encases the cell sample stream within a sheath fluid stream. This forces cells through a narrow nozzle in single file, ensuring individual interrogation. Each cell then passes through one or more focused laser beams.
When a labeled cell intersects the laser, fluorescent tags absorb energy and emit light at a longer wavelength. This emitted light, along with scattered light, is captured by sensitive detectors. Forward scatter (FSC) measures light scattered forward, providing cell size information. Side scatter (SSC) measures light scattered at 90 degrees, indicating internal complexity or granularity.
Emitted fluorescent light passes through optical filters to separate wavelengths, allowing simultaneous detection of multiple markers. This system creates a unique optical signature for each cell, providing information about its size, internal structure, and specific labeled molecules. This data is rapidly analyzed by the software, categorizing each cell based on its measured properties.
Based on this analysis, the instrument determines if a cell meets sorting criteria. After laser interrogation, the cell stream is vibrated at high frequency, breaking into individual droplets. Each droplet ideally contains one cell. If a droplet contains a cell identified for sorting, an electrical charge (positive or negative) is applied just before it detaches.
Charged droplets pass through an electrostatic deflection plate system, which generates an electric field. Positively charged droplets are deflected in one direction, while negatively charged droplets are deflected in the opposite direction. Uncharged droplets, containing cells not selected for sorting, pass straight through. This precise deflection mechanism directs the desired cells into separate collection tubes, while unwanted cells are diverted to a waste container.
Key Applications in Science and Medicine
Fluorescence Activated Cell Sorters have profoundly impacted medical research by enabling scientists to delve deeper into disease mechanisms. Researchers utilize FACS to identify specific cell types within complex tumor samples, which helps in understanding tumor heterogeneity and progression. The technology also plays a significant role in studying immune responses by characterizing different immune cell populations, such as T cells and B cells, in various infectious diseases or autoimmune conditions. Furthermore, FACS is employed to analyze stem cell populations, differentiating them based on surface markers, which is valuable for regenerative medicine and understanding developmental biology.
In clinical diagnostics, FACS provides rapid and accurate information for patient care, particularly in hematology and immunology. It is routinely used to diagnose blood cancers like leukemia and lymphoma by identifying abnormal cell populations based on their unique surface marker expression. For instance, specific patterns of CD markers (cluster of differentiation) can differentiate between various types of leukemias. The instrument also monitors immune cell counts, such as CD4+ T cell levels in HIV-infected individuals, which helps guide treatment decisions and assess disease progression.
The capabilities of FACS extend into drug discovery and development, offering efficient methods for screening potential therapeutic compounds. Researchers can use the technology to assess the effects of new drugs on specific cell populations, for example, by measuring changes in cell viability, proliferation, or protein expression. This allows for high-throughput screening of drug candidates, helping to identify promising compounds more quickly. FACS also aids in evaluating drug toxicity by detecting cellular damage or apoptosis in response to different concentrations of a substance.
Beyond medical applications, FACS has broad utility in fundamental biological studies. It is widely used for cell cycle analysis, where cells are stained with fluorescent dyes that bind to DNA, allowing researchers to quantify cells in different phases of the cell cycle (G0/G1, S, G2/M). This provides insights into cell proliferation and growth. The technology also assesses cell viability by distinguishing live cells from dead cells using membrane-impermeable dyes that only enter compromised cells. Most importantly, FACS enables the isolation of highly pure populations of specific cell types from heterogeneous samples, which are then used for subsequent experiments, such as gene expression profiling or protein analysis, ensuring reliable and accurate downstream results.