Fluorescence-Activated Cell Sorter (FACS)

A machine that reads every cell in a blood sample by its glow and hurls the ones you want into a separate tube — fifty thousand per second.

A FACS instrument takes a suspension of cells, forces them in single file through a focused laser beam, and measures the light each cell scatters and emits. Cells are labelled beforehand with fluorescent antibodies — each antibody colour marks a different surface protein or internal molecule. As each cell passes the laser, detectors record its scatter profile and up to 20–30 fluorescent colours simultaneously. A downstream droplet-charging mechanism gives the droplet containing a wanted cell a precise electric charge; deflector plates then steer it into the correct collection tube. Unwanted cells fly straight into waste. The result is a highly purified population — often >98% pure — harvested alive for downstream culture, sequencing, or transplantation.

The fluidics system wraps a slow-moving sample stream inside a faster sheath fluid (hydrodynamic focusing), so cells arrive at the laser interrogation point in single file. A piezoelectric transducer vibrates the nozzle at 30,000–90,000 Hz, breaking the stream into uniform droplets roughly 20–40 µm in diameter at the breakoff point. The electronics must predict, based on the flight time from laser to breakoff point (the 'drop delay'), exactly which droplet will contain the cell of interest. That droplet receives a brief high-voltage charge pulse; downstream deflector plates at ±200 to ±3,000 V steer it into the target collection vessel. Optical performance depends on tightly collimated spatial laser beams (typically elliptical, ~9 × 65 µm beam spot on BD Aria-class instruments) and on photomultiplier tubes or avalanche photodiodes behind bandpass filters matched to each fluorochrome emission peak.

In plain terms

Imagine sorting a million marbles by colour in under a minute. You shine a laser at each marble as it rolls past, the colour it glows tells a tiny computer what it is, and a puff of electric charge flicks it left or right before it lands in a cup. FACS does exactly that — except the 'marbles' are living cells, and the sorting happens inside a very thin stream of water.

Also called: FACS, cell sorter, flow cytometer sorter, fluorescence-activated cell sorting.

Why it matters

Isolates rare cell populations — such as haematopoietic stem cells present at 0.01% of bone marrow — in sufficient numbers for downstream functional or genomic analysis.
Index sorting links every deposited cell to its full fluorescence phenotype, enabling retrospective gating without losing the cell.
Multi-parametric data (up to 20+ colours on commercial sorters) allows the simultaneous identification of dozens of cell subsets in a single tube of blood.
Clinical applications include T-cell subset enumeration in HIV, minimal residual disease monitoring in leukaemia, and graft purification for bone-marrow transplantation.
Single-cell deposition into 96- or 384-well plates pipelines individual cells directly into whole-genome or transcriptomic sequencing workflows.

Where it lives

BD FACSAria Fusion (up to 70,000 events/sec acquisition; 6 lasers, 20 detectors)
Beckman Coulter MoFlo Astrios EQ (>100,000 events/sec acquisition; 6-way sorting)
Sony MA900 Multi-Application Cell Sorter (70,000 eps; microfluidic chip-based design)
BD FACSMelody (40,000 events/sec; simplified bench-top sorter)

Key numbers

Sort throughput: up to ~70,000 events/sec (BD FACSAria Fusion datasheet; 4-way sort purity >98% at 25k threshold rate)
Drop drive frequency: 1–100,000 Hz (range) (Piezoelectric nozzle vibration; typically 30–90 kHz during operation)
Sort purity: >98% (At 25k threshold rate, 70 psi, 90 kHz — BD datasheet)
Laser configurations: up to 6 spatially separated lasers (355, 405, 488, 532, 561, 640 nm typical; up to 51 parameters on MoFlo Astrios EQ)
Nozzle orifice: 70–130 µm (Smaller nozzle = faster drops but more cell stress; 70 µm standard for lymphocytes)

Surprising facts

A top-end cell sorter can classify and physically route more than 70,000 individual cells every second — each cell getting its own fluorescence readout, a sorting decision, and a charged droplet deflection, all in under 15 microseconds.
The droplet breakoff point is so precisely controlled that the instrument can predict which droplet a specific cell will be inside roughly 30–100 droplets before it reaches the charge electrode — a look-ahead calculation performed tens of thousands of times per second.
Modern FACS instruments measure up to 30 fluorescent parameters simultaneously on a single cell; the first published instrument (Bonner et al., 1972) measured a single parameter — the ratio of two fluorescent signals — and sorted at roughly 1,000 cells per second.
The sheath fluid stream exiting the nozzle travels at several metres per second; the entire optical interrogation of one cell — laser excitation, emission collection, and digitisation — occurs in a time window of only 1–10 microseconds.

Open questions

Sorting-induced gene expression changes: Multiple studies have reported that the mechanical and fluidic stresses of high-speed cell sorting — even under gentle conditions — transiently upregulate stress-response genes (heat-shock proteins, immediate-early genes) in sorted cells. The magnitude of the effect varies with cell type, nozzle pressure, and temperature in ways that no single biophysical model fully predicts, complicating single-cell RNA-sequencing experiments where gene expression immediately after sorting is the readout of interest.

A short history

The conceptual ancestor of FACS was Mack Fulwyler's Coulter-volume-based cell sorter, published in Science in 1965. Leonard Herzenberg at Stanford recognised that attaching fluorescence detection would make functional cell populations separable, not just cells of different sizes. Working with Bonner, Hulett, and Sweet, the Stanford group built the first fluorescence-activated cell sorter and published the foundational paper in the Review of Scientific Instruments in March 1972. Becton Dickinson commercialised the technology under a Stanford licence in the mid-1970s, coining the trademarked name 'FACS'. Multicolour capability expanded through the 1990s as more bright, spectrally distinct fluorochromes became available, and modern high-parameter instruments now resolve 30+ colours on a single cell.

Sources

Bonner et al., Rev. Sci. Instrum. (1972) — foundational FACS paper (https://pubs.aip.org/aip/rsi/article/43/3/404/305945/Fluorescence-Activated-Cell-Sorting)
BD FACSAria Fusion Technical Specifications — BD Biosciences datasheet (https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/products-pdf-folder/instruments/research-cell-sorters/instruments-tech-specs-facsaria-fusion.pdf)
Beckman Coulter MoFlo Astrios EQ product page (https://www.beckman.com/flow-cytometry/cell-sorters/moflo-astrios-eq)
Sony MA900 Multi-Application Cell Sorter specifications (https://www.sonybiotechnology.com/instruments/ma900-multi-application-cell-sorter/specifications)