Flow cytometry

Flow Cytometry

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Flow cytometry

Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric

Parametric model

Definition. A set of probability measures on indexed by a parameter is said to be a parametric model or parametric family if and only if the parameter space is a subset of ....
analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.

History
The first fluorescence-based flow cytometry device (ICP 11) was developed in the year 1968 by Wolfgang Göhde from the University of Münster, Germany (Patent No.

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Encyclopedia

Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric

Parametric model

History

The first fluorescence-based flow cytometry device (ICP 11) was developed in the year 1968 by Wolfgang Göhde from the University of Münster, Germany (Patent No. DE1815352) and first commercialized in 1968/69 by German developer and manufacturer Partec through Phywe AG in Göttingen. At that time absorption methods were still widely favored by other scientists over fluorescence methods . The original name of the flow cytometry technology was pulse cytophotometry (German: Impulscytophotometrie). Only 10 years later in 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to flow cytometry, a term which quickly became popular. Subsequently introduced flow cytometry instruments have been the Cytofluorograph (1971) from Bio/Physics Systems Inc. (later: Ortho Diagnostics), the PAS 8000 (1973) from Partec, the first FACS instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/79

Principle of flow cytometry

A beam of light

Light

Light, or visible light, is electromagnetic radiation of a wavelength that is Visible spectrum to the human eye , or up to 380?750 nm. In the broader field of physics, light is sometimes used to refer to electromagnetic radiation of all wavelengths, whether visible or not....
(usually laser

Laser

A laser is a device that emits light through a process called stimulated emission. The term laser is an acronym for light amplification by stimulated emission of radiation....
light) of a single wavelength is directed onto a hydro-dynamically focused

Hydrodynamic focusing

Hydrodynamic focusing is a technique used by microbiologists to provide more accurate results from flow cytometers or Coulter counters for determining the size of bacteria or Cell ....
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 from 0.2 to 150 micrometers 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 higher wavelength than the light source. This combination of scattered

Scattering

Scattering is a general physical process where some forms of radiation, such as light, sound, or moving particles,are forced to deviate from a straight trajectory by one or more localized non-uniformities in the medium through which they pass....
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 derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell

Cell (biology)

The cell is the structural and functional unit of all known Life organisms. It is the smallest unit of an organism that is classified as living, and is often called the building bricks of life....
volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus

Cell nucleus

In cell biology, the nucleus , also sometimes referred to as the "control center", is a membrane-enclosed organelle found in all eukaryote cell ....
, the amount and type of cytoplasm

Cytoplasm

The cytoplasm is the part of a Cell that is enclosed within the plasma membrane. In eukaryote cells the cytoplasm contains organelles, such as mitochondrion, that are filled with liquid kept separate from the rest of the cytoplasm by biological membranes....
ic granules or the membrane

Cell membrane

The cell membrane is the interface between the cellular machinery inside the cell and the fluid outside.It is a semipermeable lipid bilayer found in all cell ....
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

Microscope

A microscope is an Laboratory equipment for viewing objects that are too small to be seen by the naked or unaided eye. The science of investigating small objects using such an instrument is called microscopy....
, except that instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification

Quantification

Quantification has two distinct meanings. In mathematics and empirical science, it refers to human acts, known as counting and measuring that map human sense observations and experiences into element s of some Set of numbers....
of set parameters. To analyze solid tissues

Biological tissue

Tissue is a cellular organizational level intermediate between cells and a complete organism. Hence, a tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function....
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.
an optical system - commonly used are lamps (mercury
Mercury (element)

Mercury , also called quicksilver or hydrargyrum , is a chemical element with the symbol Hg and atomic number 80. A heavy, silvery d-block metal, mercury is one of six elements that are liquid at or near room temperature and pressure....
, xenon
Xenon

Xenon is a chemical element represented by the chemical symbol Xe. Its atomic number is 54. A colorless, heavy, odorless noble gas, xenon occurs in the Earth's atmosphere in trace amounts....
); 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) resulting in light signals.
a detector and Analogue-to-Digital Conversion (ADC) system - generating FSC and SSC as well as fluorescence signals from light into electrical signals that can be processed by a computer.
an amplification system - linear
Linear

The word linear comes from the Latin word linearis, which means created by lines.In mathematics, a linear map or function f is a function which satisfies the following two properties......
or logarithmic
Logarithmic scale

A logarithmic scale is a scale that uses the logarithm of a physical quantity instead of the quantity itself.Presentation of data on a logarithmic scale can be helpful when the data covers a large range of values – the logarithm reduces this to a more manageable range....
.
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

Phenotype

A phenotype is any observable characteristic or trait_ of an organism: such as its morphology , development, biochemical or physiological properties, or behavior....
. 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

Dimension

In mathematics, the dimension of a space is roughly defined as the minimum number of coordinates needed to specify every point within it. For example: a point on the unit circle in the plane can be specified by two Cartesian coordinates but one can make do with a single coordinate , so the circle is 1-dimensional even though it exists in...
, to produce a histogram

Histogram

In statistics, a histogram is a graphical display of tabulated frequency , shown as bars. It shows what proportion of cases fall into each of several Categorization....
, 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

Hematology, American_and_British_English_spelling_differences#Simplification_of_ae_.28.C3.A6.29_and_oe_.28.C5.93.29 haematology, is the branch of biology , pathology, clinical laboratory, internal medicine, and pediatrics that is concerned with the study of blood, the blood-forming organs, and blood diseases....
. 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. Often, data accumulated using the flow cytometer can be re-analysed (using free software e.g. WinMDI ) elsewhere freeing up the machine for other people to use.

Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting 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

Scattering

Scattering is a general physical process where some forms of radiation, such as light, sound, or moving particles,are forced to deviate from a straight trajectory by one or more localized non-uniformities in the medium through which they pass....
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 trademark

TradeMark

TradeMark is a tall, primarily residential, skyscraper in Charlotte, North Carolina. It was completed in 2007 and has 28 floors. There are 200 hundred residential units....
ed and owned by Becton Dickinson

Becton Dickinson

Becton, Dickinson and Company , is a medical technology company that manufactures and sells medical devices, instrument systems and reagents. Founded in 1897 and headquartered in Franklin Lakes, New Jersey, BD does business in nearly 50 countries with its 27,000 employees....
. While many immunologists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry. 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 and one no longer used in modern instruments. The technique was expanded by Len Herzenberg

Leonard Herzenberg

Leonard Arthur "Len" Herzenberg is an immunologist, geneticist and professor at Stanford University. His contribututions to the development of cell biology made it possible to sort viable cells by their specific properties....
who was responsible for coining the term FACS. Herzenberg won the Kyoto Prize

Kyoto Prize

The Kyoto Prize has been awarded annually since 1985 by the Inamori Foundation, founded by Kazuo Inamori. The prize is a Japanese award similar in intent to the Nobel Prize, as it recognizes outstanding works in the fields of philosophy, arts, science and technology....
in 2006 for his work in flow cytometry.

The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid

Liquid

Liquid is one of the principal states of matter. A liquid is a fluid that has the particles loose and can freely form a distinct surface at the boundaries of its bulk material....
. The flow is arranged so that there is a large separation between cells relative to their diameter

Diameter

In geometry, a diameter of a circle is any straight line segment that passes through the center of the circle and whose endpoints are on the circle....
. A vibrating

Oscillation

Oscillation is the repetitive variation, typically in time, of some measure about a central value or between two or more different states. Familiar examples include a swinging pendulum and Alternating current power....
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

Electric charge

Electric charge is a fundamental conserved property of some subatomic particles, which determines their electromagnetic interaction. Electrically charged matter is influenced by, and produces, electromagnetic fields....
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

Electrostatic deflection

Electrostatic deflection refers to a technique for modifying the path of a stream of charged particles by the use of an electric field applied transverse to the path of the particles....
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: Blue Argon Laser (488 nm) 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.

Green (usually labelled FL1): FITC
Fluorescein

Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing....
, Alexa Fluor 488, GFP
Green fluorescent protein

The green fluorescent protein is composed of 238 amino acids , originally isolated from the jellyfish Aequorea victoria that fluorescence green when exposed to blue light....
, CFSE
CFSE

CFSE is a fluorescent cell staining dye. CFSE is commonly confused with CFDA-SE, although they are not strictly the same molecule; CFDA-SE is cell permeable, while CFSE is not....
, CFDA-SE
CFDA-SE

CFDA-SE is a Cell permeable staining generally used in animal cell proliferation research. High concentrations of the dye are toxic to animal cells, however concentrations in the region of 10 concentration are typically sufficient to give strong staining with minimal cell death....
, DyLight 488
DyLight Fluor

The DyLight Fluor family of fluorescence dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. Antibodies conjugated to DyLight Dyes are produced by Thermo Fisher Scientific as well as a number of partners, including Jackson Immunoresearch, Rockland Immunochemicals, Inc., AbD Serotec,and KPL....
Orange (usually FL2): PE
Phycoerythrin

Phycoerythrin is a red protein from the light-harvesting phycobiliprotein family, present in cyanobacteria, red algae and cryptomonads.Like all phycobiliproteins, phycoerythrin is composed of a protein part, organised in a hexameric structure of alpha and beta chains, covalently binding chromophores called phycobilins....
, PI
Propidium iodide

Propidium iodide is an intercalating agent and a Fluorescence#Biochemistry and medicine molecule with a molecular mass of 668.4 Atomic mass unit that can be used to Staining DNA....
Red channel (usually FL3): PerCP, PE-Alexa Fluor 700, PE-Cy5 (TRI-COLOR), PE-Cy5.5.
Infra-red (usually FL4; not provided by all FACS machines as standard): PE-Alexa Fluor 750, PE-Cy7

Red diode laser (635 nm)

APC
Allophycocyanin

Allophycocyanin is a protein from the light-harvesting phycobiliprotein family, along with phycocyanin, phycoerythrin and phycoerythrocyanin. It is an accessory pigment to chlorophyll....
APC-Cy7
Alexa Fluor 700
Cy5
Draq-5

Violet laser (405 nm)

Pacific Orange
Amine Aqua
Pacific Blue
DAPI
DAPI

DAPI or 4',6-diamidino-2-phenylindole is a fluorescence staining that binds strongly to DNA. It is used extensively in fluorescence microscopy....
Alexa Fluor 405

Measurable parameters

This list is very long and constantly expanding.

volume and morphological
Morphology (biology)

The term morphology in biology refers to form, structure and configuration of an organism. This includes aspects of the outward appearance as well as the form and structure of the internal parts like bones and organs....
complexity of cells
cell pigment
Pigment

A pigment is a material that changes the color of light it Reflection as the result of selective color absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which the material itself emits light....
s such as chlorophyll
Chlorophyll

Chlorophyll is a green pigment found in most plants, algae, and cyanobacteria. Its name is derived from Greek language: ?????? and f????? ....
or phycoerythrin
Phycoerythrin

Phycoerythrin is a red protein from the light-harvesting phycobiliprotein family, present in cyanobacteria, red algae and cryptomonads.Like all phycobiliproteins, phycoerythrin is composed of a protein part, organised in a hexameric structure of alpha and beta chains, covalently binding chromophores called phycobilins....
DNA
DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetics instructions used in the development and functioning of all known living organisms and some viruses....
(cell cycle
Cell cycle

The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission....
analysis, cell kinetic
Kinetic

Kinetic may refer to:*Kinetic, Seiko's trademark for its automatic quartz technology.*Kinetic theory*Kinetic energy*A projectile, which is a type of kinetic weapon....
s, proliferation
Proliferation

The word proliferation can refer to:*Nuclear proliferation*Chemical weapon proliferation*Cell growth* The proliferative phase of wound healing...
etc.)
RNA
RNA

Ribonucleic acid is a type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nucleobase, a ribose sugar, and a phosphate....
chromosome
Chromosome

A chromosome is an organized structure of DNA and protein that is found in Cell . A chromosome is a single piece of DNA that contains many genes, regulatory sequence and other genetic sequence....
analysis and sorting (library construction, chromosome paint)
protein
Protein

Proteins are organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid Residue ....
expression and localization
Protein modifications, phospho-proteins
transgenic products in vivo, particularly the Green fluorescent protein
Green fluorescent protein

The green fluorescent protein is composed of 238 amino acids , originally isolated from the jellyfish Aequorea victoria that fluorescence green when exposed to blue light....
or related fluorescent * cell surface antigen
Antigen

An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The word originated from the notion that they can stimulate antibody generation....
s (Cluster of differentiation
Cluster of differentiation

The cluster of differentiation is a protocol used for the identification and investigation of cell surface molecules present on leukocytes. CD molecules can act in numerous ways, often acting as receptor or ligand important to the cell....
(CD) markers)
intracellular
Intracellular

Not to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell "....
antigens (various cytokine
Cytokine

Cytokines are a category of signaling molecules that, like hormones and neurotransmitters, are used extensively in cell communication. They are proteins, peptides or glycoproteins....
s, secondary mediators etc.)
nuclear antigen
Antigen

An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The word originated from the notion that they can stimulate antibody generation....
s
enzymatic
Enzyme

Enzymes are biomolecules that catalysis chemical reactions. Almost all enzymes are proteins. In enzymatic reactions, the molecules at the beginning of the process are called Substrate , and the enzyme converts them into different molecules, the products....
activity
pH
PH

pH is a measure of the Acid or Base of a solution. It is defined as the cologarithm of the Activity of dissolved hydrogen ions . Hydrogen ion activity coefficients cannot be measured experimentally, so they are based on theoretical calculations....
, intracellular ionized calcium
Calcium

Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft grey alkaline earth metal, and is the fifth most abundant element by mass in the earth's Crust ....
, magnesium
Magnesium

Magnesium is a chemical element with the symbol Mg, atomic number 12, atomic weight 24.3050 and common oxidation number +2.Magnesium, an alkaline earth metal, is the ninth most abundance of the chemical elements in the universe by mass....
, membrane potential
Membrane potential

Membrane potential , is the voltage difference between the interior and exterior of a cell. Because the fluid inside and outside a cell is highly conductive, whereas a cell's plasma membrane is highly resistive, the voltage change in moving from a point outside to a point inside occurs largely within the narrow width of the membrane itself...
membrane fluidity
apoptosis
Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Programmed Cell death involves a series of biochemical events leading to a characteristic cell Morphology and death, in more specific terms, a series of biochemical events that lead to a variety of morphological changes, including Bleb , changes...
(quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase
Caspase

Caspases, or cysteine-aspartic acid proteases, are a family of cysteine proteases, which play essential roles in apoptosis , necrosis and inflammation....
activity)
cell viability
Viability

Viability means in general "capacity for survival" and is more specifically used to mean a capacity for living, developing, or germinating under favorable conditions....
monitoring electropermeabilization of cells
oxidative burst
characterising multidrug resistance
Multidrug resistance

Multiple drug resistance or Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism....
(MDR) in cancer cells
glutathione
Glutathione

Glutathione is a tripeptide. It contains an unusual peptide linkage between the amino acid of cysteine and the carboxyl group of the glutamate side chain....
various combinations (DNA/surface antigens etc.)

Applications

The technology has applications in a number of fields, including molecular biology

Molecular biology

Molecular biology is the study of biology at a molecule level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry....
, pathology

Pathology

Pathology is the study and diagnosis of disease through examination of Organ , tissue , bodily fluids and whole bodies . The term also encompasses the related science study of disease processes, called General pathology....
, immunology

Immunology

Immunology is a broad branch of biomedical science science that covers the study of all aspects of the immune system in all organisms. It deals with, among other things, the physiology functioning of the immune system in states of both health and disease; malfunctions of the immune system in immunological disorders ; the physical, chemical an...
, plant biology and marine biology

Marine biology

Marine biology is the scientific study of living organisms in the ocean or other Marine or brackish bodies of water.Given that in biology many scientific classification, families and Genera have some species that live in the sea and others that live on land, marine biology classifies species based on the environment rather than on taxon...
. In the field of molecular biology it is especially useful when used with fluorescence tagged antibodies. These specific antibodies bind to antigen

Antigen

An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The word originated from the notion that they can stimulate antibody generation....
s 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

Medicine

Medicine is the art and science of healing. It encompasses a range of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
(especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting

Sperm sorting

Sperm sorting is a means of choosing what type of sperm cell is to Fertilisation the egg cell. It can be used to sort out sperm that are most healthy, as well as determination of more specific traits, such as sex selection in which spermatozoa are separated into X- and Y- chromosome bearing populations based on their difference in DNA conte...
for sex preselection). In marine biology, the auto-fluorescent properties of photosynthetic plankton

Plankton

Plankton consist of any drifting organisms that inhabit the pelagic zone of oceans, seas, or bodies of fresh water. Plankton are defined by their ecological niche rather than their Phylogenetics or taxonomy classification....
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

Yeast display

Yeast display is a technique used in the field of protein engineering. The yeast display technique was first published by the laboratory of Professor K....
and bacterial display

Bacterial display

Bacterial Display is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures ....
to identify cell surface-displayed protein variants with desired properties.

Bibliography

Flow Cytometry First Principles by Alice Longobardi Givan ISBN 0471382248
Practical Flow Cytometry by Howard M. Shapiro ISBN 0471411256
Flow Cytometry for Biotechnology by Larry A. Sklar ISBN 0195152344
Handbook of Flow Cytometry Methods by J. Paul Robinson, et al. ISBN 0471596345
Current Protocols in Cytometry, Wiley-Liss Pub.
Flow Cytometry in Clinical Diagnosis, v4, (Carey, McCoy, and Keren, eds), ASCP Press, 2007. ISBN 0891895485
Ormerod, M.G. (ed.) (2000) Flow cytometry - A practical approach. 3rd edition. Oxford University Press, Oxford, UK. ISBN 0199638241
Ormerod, M.G. (1999) Flow Cytometry. 2nd edition. Bios Scientific Publishers, Ltd. Oxford. ISBN 185996107X
Flow Cytometry - a Basic Introduction. Michael G. Ormerod, 2008. ISBN 978-0955981203

External links

- the Flow Informatics and Computation Cytometry Society
Image Cells in Flow.

Flow cytometry

History

Principle of flow cytometry

Flow cytometers

Fluorescence-Activated Cell Sorting

Fluorescent labels

Measurable parameters

Applications

See also

Bibliography

External links