|
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 frequency (colour) 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 in 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.
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.
Flow cytometers can also be configured as sorting instruments (fluorescent-activated cell sorting or FACS). It should be noted however, that "FACS" is not a generic term and should not be used to describe cell sorting since the term is trademarked by Becton-Dickinson. As cells or particles pass through the instrument they can be selectively charged, based on user defined parameters, and can be deflected into separate paths of flow directed to different collection tubes. It is therefore possible to separate up to 4 defined populations of cells from an original mix with a high degree of accuracy and speed (up to ~90,000 cells per second in theory).
The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two dimensional dot plots or in even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by a 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 dye's emission spectra overlap [1], signals at the detectors have to be compensated electronically as well as computationally.
Major manufacturers
- Beckman Coulter (ex-Coulter): Cytomics FC500/FC500-MPL, Cell Lab Quanta SC, Cell Lab Vi-Cell, Epics XL/XL-MCL; Epics Altra (Hypersort) (PC platform)
- BD Biosciences: (FACS): FACSCalibur, FACScan, FACSort, FACSVantage (Mac OS platform) FACSCanto, BD LSR II, FACSarray, FACSAria, FACSDiVa (PC Platform)
Specialty instruments
Some manufactures have focused on developing specialized flow cytometers.
- Amnis: ImageStream imaging flow cytometer (PC Platform) [2]
- Bay bioscience corp: JSAN (PC platform) [3]
- CytoBuoy : an instrument specialized for oceanographic applications [4]
- Cytopeia: Influx (PC platform)
- Dako (ex-Dako Cytomation): MoFlo, Cyan (IBM-PC platform) [5]
- Fluid Imaging Technologies: FlowCAM imaging flow cytometer and VisualSpreadsheet analysis software (PC platform)
- Guava Technologies: Personal Cell Analysis (PCA) System, Easycyte, Easycyte mini, PCA-96 (PC platform) [6]
- Partec (for a period associated with Dako): PAS; CyFlow; CCA; PA (PC platform) [7]
- PointCare Technologies: AuRICA [8]
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
- 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 multi-drug resistance (MDR) in cancer cells
- glutathione
- various combinations (DNA/surface antigens etc.)
Applications
Use of flow cytometry in oceanography
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, heamatology, tumor immunology and chemotherapy, genetics). 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.
See also
External links
Resources
Professional societies
Laboratories
|
