Fluorescent-activated Cell Sorting
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Flow
cytometry Cytometry is the measurement of number and characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or a ...
(FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer. Flow cytometry is routinely used in basic research, clinical practice, and
clinical trial Clinical trials are prospective biomedical or behavioral research studies on human subject research, human participants designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel v ...
s. Uses for flow cytometry include: * Cell counting *
Cell sorting Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intra ...
* Determining cell characteristics and function * Detecting
microorganism A microorganism, or microbe,, ''mikros'', "small") and ''organism'' from the el, ὀργανισμός, ''organismós'', "organism"). It is usually written as a single word but is sometimes hyphenated (''micro-organism''), especially in olde ...
s *
Biomarker In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
detection *
Protein engineering Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to im ...
detection * Diagnosis of health disorders such as blood cancers * Measuring genome size A flow cytometry analyzer is an instrument that provides quantifiable data from a sample. Other instruments using flow cytometry include cell sorters which physically separate and thereby purify cells of interest based on their optical properties.


History

The first impedance-based flow cytometry device, using the Coulter principle, was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter. Mack Fulwyler was the inventor of the forerunner to today's flow cytometers - particularly the cell sorter. Fulwyler developed this in 1965 with his publication in ''
Science Science is a systematic endeavor that Scientific method, builds and organizes knowledge in the form of Testability, testable explanations and predictions about the universe. Science may be as old as the human species, and some of the earli ...
''. The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the
University of Münster The University of Münster (german: Westfälische Wilhelms-Universität Münster, WWU) is a public research university located in the city of Münster, North Rhine-Westphalia in Germany. With more than 43,000 students and over 120 fields of stud ...
, filed for patent on 18 December 1968 and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in
Göttingen Göttingen (, , ; nds, Chöttingen) is a college town, university city in Lower Saxony, central Germany, the Capital (political), capital of Göttingen (district), the eponymous district. The River Leine runs through it. At the end of 2019, t ...
. At that time, absorption methods were still widely favored by other scientists over
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
methods. Soon after, flow cytometry instruments were developed, including the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS (fluorescence-activated cell sorting) instrument from
Becton Dickinson Becton, Dickinson and Company, also known as BD, is an American multinational medical technology company that manufactures and sells medical devices, instrument systems, and reagents. BD also provides consulting and analytics services in certai ...
(1974), the ICP 22 (1975) from Partec/Phywe and the Epics from
Coulter Coulter may refer to: People * Coulter (surname) * Coulter Osborne (born 1934), Canadian arbitrator and former Associate Chief Justice of Ontario Places * Coulter, South Lanarkshire, Scotland, a village and civil parish * Coulter, Iowa, United Sta ...
(1977/78). The first label-free high-frequency impedance flow cytometer based on a patented microfluidic "lab-on-chip", Ampha Z30, was introduced by Amphasys (2012).


Name of the technology

The original name of the fluorescence-based flow cytometry technology was "pulse cytophotometry" (
German German(s) may refer to: * Germany (of or related to) **Germania (historical use) * Germans, citizens of Germany, people of German ancestry, or native speakers of the German language ** For citizens of Germany, see also German nationality law **Ger ...
: ''Impulszytophotometrie''), based on the first patent application on fluorescence-based flow cytometry. At the 5th American Engineering Foundation Conference on Automated Cytology in Pensacola (Florida) in 1976 - eight years after the introduction of the first fluorescence-based flow cytometer (1968) - it was agreed to commonly use the name "flow cytometry", a term that quickly became popular.


Flow cytometers

Modern flow cytometers are able to analyze many thousands of particles per second, in "real time" and, if configured as cell sorters, can actively separate and isolate particles with specified optical properties at similar rates. A flow cytometer is similar to a
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisibl ...
, except that, instead of producing an image of the cell, flow cytometry offers high-throughput, automated quantification of specified optical parameters on a cell-by-cell basis. To analyze solid tissues, a single-cell suspension must first be prepared. A flow cytometer has five main components: a flow cell, a measuring system, a detector, an amplification system, and a computer for analysis of the signals. The flow cell has a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. The measuring system commonly uses measurement of impedance (or conductivity) and optical systems - lamps ( mercury,
xenon Xenon is a chemical element with the symbol Xe and atomic number 54. It is a dense, colorless, odorless noble gas found in Earth's atmosphere in trace amounts. Although generally unreactive, it can undergo a few chemical reactions such as the ...
); high-power water-cooled lasers (
argon Argon is a chemical element with the symbol Ar and atomic number 18. It is in group 18 of the periodic table and is a noble gas. Argon is the third-most abundant gas in Earth's atmosphere, at 0.934% (9340 ppmv). It is more than twice as a ...
,
krypton Krypton (from grc, κρυπτός, translit=kryptos 'the hidden one') is a chemical element with the symbol Kr and atomic number 36. It is a colorless, odorless, tasteless noble gas that occurs in trace amounts in the atmosphere and is often ...
, dye laser); low-power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet) resulting in light signals. The detector and analog-to-digital conversion (ADC) system converts analog measurements of forward-scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence signals into digital signals that can be processed by a computer. The amplification system can be
linear Linearity is the property of a mathematical relationship ('' function'') that can be graphically represented as a straight line. Linearity is closely related to '' proportionality''. Examples in physics include rectilinear motion, the linear ...
or
logarithmic Logarithmic can refer to: * Logarithm, a transcendental function in mathematics * Logarithmic scale, the use of the logarithmic function to describe measurements * Logarithmic spiral, * Logarithmic growth * Logarithmic distribution, a discrete pr ...
. The process of collecting data from samples using the flow cytometer is termed "acquisition". Acquisition is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer. The software is capable of adjusting parameters (e.g., voltage, compensation) for the sample being tested, and also assists in displaying initial sample information while acquiring sample data to ensure that parameters are set correctly. Early flow cytometers were, in general, experimental devices, but technological advances have enabled widespread applications for use in a variety of both clinical and research purposes. Due to these developments, a considerable market for instrumentation, analysis software, as well as the reagents used in acquisition such as fluorescently labeled antibodies have been developed. Modern instruments usually have multiple lasers and fluorescence detectors. The current record for a commercial instrument is ten lasers and 30 fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their
phenotypic In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
markers. 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.


Hardware


Fluidics system of a flow cytometer

Cells must pass uniformly through the center of focused laser beams to accurately measure optical properties of cells in any flow cytometer. The purpose of the fluidic system is to move the cells one by one through the lasers beam and throughout the instrument. Fluidics in a flow cytometer with cell sorting capabilities also use the stream to carry sorted cells into collection tubes or wells.


Hydrodynamic focusing

For precise positioning of cells in a liquid jet, hydrodynamic focusing is used in most cytometers. The cells in suspension enter into the instrument enclosed by an outer sheath fluid. The sample core is maintained in the center of the sheath fluid. The sample input rate or how fast the cells flow through to the laser interrogation can be controlled by the pressure of the sheath fluid on the sample core. Under optimal conditions, the central fluid stream and sheath fluid do not mix.


Acoustic-assisted hydrodynamic focusing

Acoustic focusing technology is used in some flow cytometers to support hydrodynamic focusing. Acoustic waves (>2 MHz) pre-focus the sample before introduction to sheath fluid. The pre-focused sample is then injected into the hydrodynamic core and flowed through the instrument. This may help with increasing data accuracy under high sample input rates.


Optics and electronics


Optical filters

Light emitted from
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with ...
s are in a spectrum of wavelengths, so combining multiple fluorophores may cause overlap. To add specificity,
optical filter An optical filter is a device that selectively transmits light of different wavelengths, usually implemented as a glass plane or plastic device in the optical path, which are either dyed in the bulk or have interference coatings. The optic ...
s and dichroic mirrors are used to filter and move light to the detectors such as photomultiplier tubes (PMTs) or
avalanche photodiodes An avalanche photodiode (APD) is a highly sensitive semiconductor photodiode detector that exploits the photoelectric effect to convert light into electricity. From a functional standpoint, they can be regarded as the semiconductor analog of pho ...
(APD). Optical filters are designed as band pass (BP), long pass (LP), or short pass (SP) filters. Most flow cytometers uses dichroic mirrors and band pass filters to select specific bands of the optical spectrum.


Prisms, gratings, and spectral flow cytometry

Spectral flow cytometry uses prisms or
diffraction grating In optics, a diffraction grating is an optical component with a periodic structure that diffracts light into several beams travelling in different directions (i.e., different diffraction angles). The emerging coloration is a form of structural ...
s to disperse the emitted light of a marker across a detector array. This allows for the full spectra from each particle to be measured. The measured spectra from single cells are subsequently unmixed by using reference spectra of all used dyes and the autofluorescence spectrum. This may allow for a wider panel design and the application of new biological markers.


Imaging flow cytometry

Imaging flow cytometry (IFC) captures multichannel images of cells. Detectors used in imaging platforms can be equipped with
charge-coupled device A charge-coupled device (CCD) is an integrated circuit containing an array of linked, or coupled, capacitors. Under the control of an external circuit, each capacitor can transfer its electric charge to a neighboring capacitor. CCD sensors are ...
(CCD) or complementary metal-oxide-semiconductor (CMOS) to capture images of individual cells.


Data analysis


Compensation

Each fluorochrome has a broad fluorescence spectrum. When more than one fluorochrome is used, the overlap between fluorochromes can occur. This situation is called spectrum overlap. This situation needs to be overcome. For example, the emission spectrum for FITC and PE is that the light emitted by the fluorescein overlaps the same wavelength as it passes through the filter used for PE. This spectral overlap is corrected by removing a portion of the FITC signal from the PE signals or vice versa. This process is called color compensation, which calculates a fluorochrome as a percentage to measure itself. Compensation is the mathematical process by which spectral overlap of multiparameter flow cytometric data is corrected. Since fluorochromes can have wide-ranging spectrum, they can overlap, causing the undesirable result of confusion during the analysis of data. This overlap, known as spillover and quantified in the spillover coefficient, is usually caused by detectors for a certain fluorochrome measuring a significant peak in wavelength from a different fluorochrome. Linear algebra is most often used to make this correction. In general, when graphs of one or more parameters are displayed, it is to show that the other parameters do not contribute to the distribution shown. Especially when using the parameters which are more than double, this problem is more severe. Currently, no tools have been discovered to efficiently display multidimensional parameters. Compensation is very important to see the distinction between cells.


Gating

The data generated by flow-cytometers can be plotted in a single
dimension In physics and mathematics, the dimension of a mathematical space (or object) is informally defined as the minimum number of coordinates needed to specify any point within it. Thus, a line has a dimension of one (1D) because only one coor ...
, to produce a
histogram A histogram is an approximate representation of the distribution of numerical data. The term was first introduced by Karl Pearson. To construct a histogram, the first step is to " bin" (or " bucket") the range of values—that is, divide the ent ...
, 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
hematology Hematology ( always spelled haematology in British English) is the branch of medicine concerned with the study of the cause, prognosis, treatment, and prevention of diseases related to blood. It involves treating diseases that affect the pro ...
. Individual single cells are often distinguished from cell doublets or higher aggregates by their "time-of-flight" (denoted also as a "pulse-width") through the narrowly focused laser beam 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. Data accumulated using the flow cytometer can be analyzed using software. Once the data is collected, there is no need to stay connected to the flow cytometer and analysis is most often performed on a separate computer. This is especially necessary in core facilities where usage of these machines is in high demand.


Computational analysis

Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK in Immunology Database and Analysis Portal (ImmPort), SamSPECTRAL and flowClust in Bioconductor, and FLAME in GenePattern. T-Distributed Stochastic Neighbor Embedding (tSNE) is an algorithm designed to perform dimensionality reduction, to allow visualization of complex multi-dimensional data in a two-dimensional "map". Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods,) to provide an objective way to compare and evaluate the flow cytometry data clustering methods, and also to establish guidance about appropriate use and application of these methods.


FMO controls

Fluorescence minus one (FMO) controls are important for data interpretation when building multi-color panels - in which a cell is stained with multiple fluorochromes simultaneously. FMO controls provide a measure of fluorescence spillover in a given channel and allow for compensation. To generate a FMO control, a sample is stained with all the fluorochromes except the one that is being tested - meaning if you are using 4 different fluorochromes your FMO control must contain only 3 of them (example: fluorochromes - A, B, C, D; FMOs - ABC_, AB_D, A_CD, _BCD).


Cell sorting by flow cytometry

Cell sorting Cell sorting is the process through which a particular cell type is separated from others contained in a sample on the basis of its physical or biological properties, such as size, morphological parameters, viability and both extracellular and intra ...
is a method to purify cell populations based on the presence or absence of specific physical characteristics. In flow cytometers with sorting capabilities, the instrument detects cells using parameters including cell size, morphology, and protein expression, and then droplet technology to sort cells and recover the subsets for post-experimental use. The first prototype sorter was built at the
Los Alamos National Laboratory Los Alamos National Laboratory (often shortened as Los Alamos and LANL) is one of the sixteen research and development laboratories of the United States Department of Energy (DOE), located a short distance northwest of Santa Fe, New Mexico, i ...
(LANL) in 1965 by physicist Mack J. Fulwyler by joining a Coulter volume sensor with the newly invented ink jet printer. Live cell cell sorter or fluorescence-activated cell sorter (FACS) was generated by Len Herzenberg, who subsequently won the Kyoto Prize in 2006 for his seminal work. Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers. The collection process starts when a sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts. The stream then carries the cell through a vibrating nozzle which generates droplets with most containing either one cell or no cells. An electrical charging ring is placed just at the point where the stream breaks into droplets and a charge is placed on the ring based immediately prior to fluorescence intensity being measured; the opposite charge is trapped on the droplet as it breaks from the stream and the droplets are therefore charged. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based on 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. After collecting, these cells can be further cultured, manipulated, and studied.


Labels

Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties. Labels, dyes, and stains can be used for multi-parametric analysis (understand more properties about a cell).
Immunophenotyping Immunophenotyping is a technique used to study the protein expressed by cells. This technique is commonly used in basic science research and laboratory diagnostic purpose. This can be done on tissue section (fresh or fixed tissue), cell suspension, ...
is the analysis of heterogeneous populations of cells using labeled antibodies and other fluorophore containing reagents such as dyes and stains.


Fluorescent labels

A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores, or simply "fluors", are typically attached to an antibody that recognizes a target feature on or in the cell; they may also be attached to a chemical entity with affinity for the
cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (the ...
or another cellular structure. Each fluorophore has a characteristic peak
excitation Excitation, excite, exciting, or excitement may refer to: * Excitation (magnetic), provided with an electrical generator or alternator * Excite Ballpark, located in San Jose, California * Excite (web portal), web portal owned by IAC * Electron ...
and
emission Emission may refer to: Chemical products * Emission of air pollutants, notably: **Flue gas, gas exiting to the atmosphere via a flue ** Exhaust gas, flue gas generated by fuel combustion ** Emission of greenhouse gases, which absorb and emit radi ...
wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. The maximum number of distinguishable fluorescent labels is thought to be 17 or 18, and this level of complexity necessitates laborious optimization to limit artifacts, as well as complex
deconvolution In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a de ...
algorithms to separate overlapping spectra. Flow cytometry uses fluorescence as a quantitative tool; the utmost sensitivity of flow cytometry is unmatched by other fluorescent detection platforms such as
confocal microscopy Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a s ...
. Absolute fluorescence sensitivity is generally lower in
confocal microscopy Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a s ...
because out-of-focus signals are rejected by the confocal optical system and because the image is built up serially from individual measurements at every location across the cell, reducing the amount of time available to collect signal.


Quantum dots

Quantum dot Quantum dots (QDs) are semiconductor particles a few nanometres in size, having optical and electronic properties that differ from those of larger particles as a result of quantum mechanics. They are a central topic in nanotechnology. When the q ...
s are sometimes used in place of traditional fluorophores because of their narrower emission peaks.


Isotope labeling

Mass cytometry overcomes the fluorescent labeling limit by utilizing
lanthanide The lanthanide () or lanthanoid () series of chemical elements comprises the 15 metallic chemical elements with atomic numbers 57–71, from lanthanum through lutetium. These elements, along with the chemically similar elements scandium and y ...
isotopes attached to antibodies. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for 30 labels. Mass cytometry is fundamentally different from flow cytometry: cells are introduced into a plasma, ionized, and associated isotopes are quantified via
time-of-flight mass spectrometry Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined by a time of flight measurement. Ions are accelerated by an electric field of known strength. This acceleration res ...
. Although this method permits the use of a large number of labels, it currently has lower throughput capacity than flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting.


Cytometric bead array

In addition to the ability to label and identify individual cells via fluorescent antibodies, cellular products such as cytokines, proteins, and other factors may be measured as well. Similar to
ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
sandwich assays, cytometric bead array ( CBA) assays use multiple bead populations typically differentiated by size and different levels of fluorescence intensity to distinguish multiple analytes in a single assay. The amount of the analyte captured is detected via a biotinylated antibody against a secondary epitope of the protein, followed by a streptavidin-R-phycoerythrin treatment. The fluorescent intensity of R-phycoerythrin on the beads is quantified on a flow cytometer equipped with a 488 nm excitation source. Concentrations of a protein of interest in the samples can be obtained by comparing the fluorescent signals to those of a standard curve generated from a serial dilution of a known concentration of the analyte. Commonly also referred to as cytokine bead array (CBA).


Impedance flow cytometry

Impedance-based single cell analysis systems are commonly known as Coulter counters. They represent a well-established method for counting and sizing virtually any kind of cells and particles. The label-free technology has recently been enhanced by a " lab-on-a-chip" based approach and by applying high frequency
alternating current Alternating current (AC) is an electric current which periodically reverses direction and changes its magnitude continuously with time in contrast to direct current (DC) which flows only in one direction. Alternating current is the form in which ...
(AC) in the radio frequency range (from 100 kHz to 30 MHz) instead of a static
direct current Direct current (DC) is one-directional flow of electric charge. An electrochemical cell is a prime example of DC power. Direct current may flow through a conductor such as a wire, but can also flow through semiconductors, insulators, or eve ...
(DC) or low frequency AC field. This patented technology allows a highly accurate cell analysis and provides additional information like membrane
capacitance Capacitance is the capability of a material object or device to store electric charge. It is measured by the change in charge in response to a difference in electric potential, expressed as the ratio of those quantities. Commonly recognized a ...
and
viability Viability is the ability of a thing (a living organism, an artificial system, an idea, etc.) to maintain itself or recover its potentialities. Viability or viable may refer to: Biology, medicine or ecology * Viability selection, the selection of ...
. The relatively small size and robustness allow battery powered on-site use in the field.


Measurable parameters

* Apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes,
caspase Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cyst ...
activity) * Cell adherence (for instance, pathogen-host cell adherence) * Cell
pigment A pigment is a colored material that is completely or nearly insoluble in water. In contrast, dyes are typically soluble, at least at some stage in their use. Generally dyes are often organic compounds whereas pigments are often inorganic comp ...
s such as
chlorophyll Chlorophyll (also chlorophyl) is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words , ("pale green") and , ("leaf"). Chlorophyll allow plants to ...
or
phycoerythrin Phycoerythrin (PE) is a red protein-pigment complex from the light-harvesting phycobiliprotein family, present in cyanobacteria, red algae and cryptophytes, accessory to the main chlorophyll pigments responsible for photosynthesis.The red pigment ...
* Cell surface
antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
s (
Cluster of differentiation The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophen ...
(CD) markers) * Cell
viability Viability is the ability of a thing (a living organism, an artificial system, an idea, etc.) to maintain itself or recover its potentialities. Viability or viable may refer to: Biology, medicine or ecology * Viability selection, the selection of ...
* Circulating tumor cells: isolation and purification * Characterising
multidrug resistance Multiple drug resistance (MDR), multidrug resistance or multiresistance is antimicrobial resistance shown by a species of microorganism to at least one antimicrobial drug in three or more antimicrobial categories. Antimicrobial categories a ...
(MDR) in cancer cells * Chromosome analysis and sorting (library construction, chromosome paint) * DNA copy number variation (by Flow-FISH or BACs-on-Beads technology) * Enzymatic activity *
Glutathione Glutathione (GSH, ) is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources such as reactive oxygen species, free radicals, pe ...
*
Intracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...
antigens (various
cytokine Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in a ...
s, secondary mediators, etc.) * Membrane fluidity * Monitoring electropermeabilization of cells * Nuclear
antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
s * Oxidative burst * pH, intracellular ionized
calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar t ...
,
magnesium Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ...
,
membrane potential Membrane potential (also transmembrane potential or membrane voltage) is the difference in electric potential between the interior and the exterior of a biological cell. That is, there is a difference in the energy required for electric charge ...
*
Protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...
expression and localization * Protein modifications, phospho-proteins * Scattering of light can be used to measure volume (by forward scatter) and morphological complexity (by side scatter) of cells or other particles, even those that are non-fluorescent. These are conventionally abbreviated as FSC and SSC respectively. * Total DNA content (
cell cycle analysis Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent ...
, cell kinetics,
proliferation Proliferation may refer to: Weapons *Nuclear proliferation, the spread of nuclear weapons, material, and technology *Chemical weapon proliferation, the spread of chemical weapons, material, and technology * Small arms proliferation, the spread of ...
,
ploidy Ploidy () is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respective ...
,
aneuploidy Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include a difference of one or more complete sets of chromosomes. A cell with a ...
, endoreduplication, etc.) * Total
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
content * Transgenic products ''in vivo'', particularly the
green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aeq ...
or related fluorescent proteins * Various combinations (DNA/surface antigens, etc.)


Applications

The technology has applications in a number of fields, including
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
,
pathology Pathology is the study of the causes and effects of disease or injury. The word ''pathology'' also refers to the study of disease in general, incorporating a wide range of biology research fields and medical practices. However, when used in ...
,
immunology Immunology is a branch of medicineImmunology for Medical Students, Roderick Nairn, Matthew Helbert, Mosby, 2007 and biology that covers the medical study of immune systems in humans, animals, plants and sapient species. In such we can see ther ...
, virology, plant biology and
marine biology Marine biology is the scientific study of the biology of marine life, organisms in the sea. Given that in biology many phyla, families and genera have some species that live in the sea and others that live on land, marine biology classifie ...
. It has broad application in
medicine Medicine is the science and Praxis (process), practice of caring for a patient, managing the diagnosis, prognosis, Preventive medicine, prevention, therapy, treatment, Palliative care, palliation of their injury or disease, and Health promotion ...
especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for
sex preselection Sex selection is the attempt to control the sex of the offspring to achieve a desired sex. It can be accomplished in several ways, both pre- and post-implantation of an embryo, as well as at childbirth. It has been marketed under the title family ...
. Flow cytometry is widely applied to detect sperm cells abnormality associated with
DNA fragmentation DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually a ...
in male fertility assays. Also, it is extensively used in research for the detection of
DNA damage DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...
, caspase cleavage and apoptosis. Photoacoustic flow cytometry is used in the study of multi-drug-resistant bacteria (most commonly MRSA) to detect, differentiate, and quantify bacteria in the blood marked with dyed bacteriophages. In
neuroscience Neuroscience is the science, scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions and disorders. It is a Multidisciplinary approach, multidisciplinary science that combines physiology, an ...
, co-expression of cell surface and intracellular antigens can also be analyzed. In microbiology, it can be used to screen and sort transposon mutant libraries constructed with a GFP-encoding transposon (TnMHA), or to assess viability. 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. The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher price and a slower pace.


Aquatic research

In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains. This research started in 1981 when
Clarice Yentsch Clarice Morel Yentsch is a scientist, author, education and museum professional, and community benefactor. As a scientist, she pioneered the use of flow cytometry to investigate marine phytoplankton and co-founded Bigelow Laboratory for Ocean Scien ...
used flow cytometry to measure the fluorescence in a red tide producing dinoflagellate. The next year researchers published flow cytometric measurements of multiple algal species which could be distinguished based on their fluorescence characteristics. By 1983, marine researchers were assembling their own flow cytometers or using commercially available flow cytometers on seawater samples collected off Bermuda to demonstrate that phytoplankton cells could be distinguished from non-living material and that cyanobacteria could be sorted from a mixed community and subsequently cultured in the lab. Flow cytometry also allowed marine researchers to distinguish between dimly-fluorescing '' Prochlorococcus'' and heterotrophic microorganisms, a distinction that is difficult with microscopy-based assessments. Advances in technology now allow aquatic scientists to use flow cytometers continuously during research cruises and flow cytometers are used to provide images of individual phytoplankton cells. Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity, to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity, and to measure biogeochemical rates of multiple processes in the ocean.


Cell proliferation assay

Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments. This dye binds covalently with the long-lived molecules present inside the cell. When the cells divide, the molecules divide too and, the daughter cells possess half the dye than the parent population. This decrease in the intensity can be visualized by flow cytometry. In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines.


Measuring genome size

Flow cytometry has been used to measure genome sizes, or more precisely: the amount of DNA in a cell or
nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom * Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucl ...
. Although genomes can be analyzed with more precision by
genome sequencing Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...
, this is often difficult due to a high fraction of micro-chromosomes or repetitive sequences which may be missed by sequencing (or which get filtered out during the analysis step when they cannot be assigned to
chromosomes A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
). However, flow cytometry is not perfect either. The resulting genome sizes may differ based on the dye used. An analysis of fish genomes resulted in significantly different genome sizes when
propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a flu ...
(PI) and DAPI were used, respectively. For instance, the genome of '' Anguilla japonica'' was found to contain 1.09 pg of DNA with PI vs. 1.25 pg with DAPI. Similarly, the genome of '' Myxocyprinus asiaticus'' was found to contain 2.75 pg of DNA (PI) vs. 3.08 pg (DAPI). That is, the differences were on the order of 12-14%.


See also

*
Annexin A5 affinity assay In molecular biology, an annexin A5 affinity assay is a test to quantify the number of cells undergoing apoptosis. The assay uses the protein annexin A5 to tag apoptotic and dead cells, and the numbers are then counted using either flow cytometr ...
, a test for cells undergoing apoptosis, often uses flow cytometry *
Cell cycle analysis Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent ...
* Coulter counter *
Cytometry Cytometry is the measurement of number and characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or a ...
* Dielectrophoresis * Flow Cytometry Standard *
Mass cytometry Mass cytometry is a mass spectrometry technique based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry used for the determination of the properties of cells (cytometry). In this approach, antibodies are conjug ...
* Microfluorimetry *
Viability assay A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover a state of survival. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quan ...


Notes


References


Further reading

* * * * * * * * * * * *


External links

* * {{Authority control Blood tests Cell biology Clinical pathology Laboratory techniques Medical tests