Flow cytometry

Overview
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.

Principle
A beam of light (usually laser light) of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to extrapolate various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.

Flow cytometers
Modern flow cytometers are able to analyse several thousand particles every second, in "real time", and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification of set parameters. To analyze solid tissues single-cell suspension must first be prepared.

A flow cytometer has 5 main components:
 * a flow cell - liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing.
 * a light source - commonly used are lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet).
 * a detector and Analogue to Digital Conversion (ADC) system - generating FSC and SSC as well as fluorescence signals.
 * an amplification system - linear or logarithmic.
 * a computer for analysis of the signals.

Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as fluorescently-labeled antibodies and analysis software.

Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a commercial instrument is 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and detectors allows for multiple antibody labelling, and can more precisely identify a target population by their phenotype. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells.

The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates". Specific gating protocols exist for diagnostic and clinical purposes especially in relation to haematology. The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally.

Fluorescence-activated cell sorting
Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The acronym FACS is trademarked and owned by Becton Dickinson although it is used within the scientific community as a general term. The first cell sorter was invented by Mack Fulwyler in 1965 using the principle of Coulter volume, a relatively difficult technique to use for sorting. The technique was expanded by Len Herzenberg who was responsible for the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow cytometry.

The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.

Fluorescent labels
The fluorescence labels that can be used, will depend on the lamp or laser used to excite the fluorochromes and on the detectors available: This is an air cooled laser and therefore cheaper to set up and run. It is the most commonly available laser on single laser machines.
 * blue argon laser (488 nm)
 * Green (usually labelled FL1): FITC, GFP, CFSE, CFDA-SE
 * Orange (usually FL2): PE
 * Red channel (FL3): PerCP, PE-Cy5, PE-Cy5.5, PI
 * Infra-red (FL4): PE-Cy7


 * UV helium-cadmium (HeCd) laser (325 nm)
 * Ultraviolet: Hydroxycoumarin
 * Blue: Y66H, Hoechst 33342
 * Green: Y66F


 * UV (HBO) lamp (366 nm) (usually used for DNA work)
 * Violet: Indo-1
 * Blue: DAPI, Hoechst 33342, AMCA
 * Blue-green: MBB


 * violet diode laser (405 nm)
 * Pacific Blue® (manufactured by BD)
 * AmCyan

Seldom used because the colour is too close to the 488nm blue laser that most machines use.
 * green helium-neon (HeNe) laser (543nm)


 * red helium-neon (HeNe) laser (633 nm) or red diode laser (635 nm)
 * APC
 * APC-Cy7
 * Cy5

Measurable parameters
This list is very long and constantly expanding.
 * volume and morphological complexity of cells
 * cell pigments such as chlorophyll or phycoerythrin
 * DNA (cell cycle analysis, cell kinetics, proliferation etc.)
 * RNA
 * chromosome analysis and sorting (library construction, chromosome paint)
 * protein expression and localization
 * transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent proteins
 * cell surface antigens (Cluster of differentiation (CD) markers)
 * intracellular antigens (various cytokines, secondary mediators etc.)
 * nuclear antigens
 * enzymatic activity
 * pH, intracellular ionized calcium, magnesium, membrane potential
 * membrane fluidity
 * apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity)
 * cell viability
 * monitoring electropermeabilization of cells
 * oxidative burst
 * characterising multidrug resistance (MDR) in cancer cells
 * glutathione
 * various combinations (DNA/surface antigens etc.)

Applications
The technology has applications in a number of fields, including molecular biology, pathology, immunology, plant biology and marine biology. In the field of molecular biology it is especially useful when used with fluorescence tagged antibodies. These specific antibodies bind to antigens on the target cells and help to give information on specific characteristics of the cells being studied in the cytometer. It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting in IVF). In marine biology, the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties.